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Cellulase Enzymes for the Conversion of Biomass to Biofuels and Chemicals

Improvements to Saccharification Enzymes allow for a faster, more stable and more economical process for cellulose breakdown.

National Renewable Energy Laboratory

Contact NREL About This Technology



Technology Marketing SummaryAll plant matter on earth consists of long insoluable chains of covalently bonded glucose molecules known as cellulose. Cellulose must first be broken down into component sugars in a process known as saccharification before fermentation can convert these carbohydrates into fuel alcohols. Saccharification requires three sequential cellulase enzymes (endoglucanases, exoglucanases, and cellobiases) in which the product of one enzyme becomes the substrate for the next enzyme. The problem with this process is these enzymes are often self inhibited by their products or degrade at high temperatures. These limiting factors can make biofuels very expensive and unable to compete with petroleum derived fuels. Scientists at NREL have made modifications to several saccharification enzymes in order to improve thermostability, specific activity, inhibition reduction, and most importantly cut costs. Description1. E1 - Endoglucanases speed up the conversion process by making more active sites for subsequent enzymes (specifically CBH1). Endoglucanases have a cleft active site, allowing them to cleave cellulose at random sites along the hydrocarbon chain – resulting in shorter cellulose chains, more chains, and more reducing ends for CBH1 to act on.
ROI 89-05: NREL’s endoglucanase (E1) is derived from the thermo stable bacteria Acidothermus cellulolyticus extracted from a hot pool in Yellowstone, rather than the mesophilic cellulases used in industry. NREL’s endoglucanase posseses a high degree of thermostability, operates at a high optimum temperature, and has a high threshold for inactivation. NREL’s 89-05 discloses the method for culturing and purifying this strain from Acidothermus cellulolyticus. (others 95-56, 94-08, 93-92, 89-05CIP)

2. CBH1 – Cellobiohydrolases are exoglucanases derived from fungi. CBH1 is made up of a carbohydrate binding site, a linker region and a catalytic domain. The carbohydrate binding site can only bind to reducing ends of cellulose, which is why E1 and CBH1 work so well sequentially. Once the binding site grabs the cellulose chain, it is strung through tunnel-shaped active site where the cellulose is broken down into two-sugar segments called cellobiose.

99-45: With the objective of creating a ternary cellulase system hosting E1, CBH1, and BGL - NREL scientists disclose a method to create reduced glycosylation variants of CBH1 from T. reesei, enabling it to be inserted and activated in a heterologous host. Also disclosed are amino acid mutations and helix capping mutations which improve thermal tolerance and improved performance.

03-05: When NREL scientists formulated CBH1 derived from the fungus Penicillium funiculosum with a small amount of endoglucanase from Acidothermus cellulolyticus and expressed it in an Aspergillus host, they found specific activity to increase by over 200% when compared to a cellobiohydrolase from T. ressei.

3. BGL- Beta-glucosidases are the cellobiases that complete the last step of the saccharification process by cleaving the cellobiose into its two subsequent glucoses. Glucose is a fermentable sugar which is then fermented into alcohols (mainly, ethanol).

93-21: NREL’s BGL from Acidothermus cellulolyticus has a high degree of activity and thermostability.

4. Alternative Biomass Conversion Enzymes
There is a need to generate alternative enzymes capable of assisting in the commercial-scale processing of cellulose and hemicellulose to sugar for use in biofuel production. NREL has developed the following cellulases:

01-37: Gux A, a novel thermostable cellulase with two catalytic domains, one from GH6 and one from GH12, could potentially have both exoglucanase and endoglucanase activity.

98-37: Dex1, an alkaline tolerant dextranase to break down dextran, an extracellular carbohydrate storage compound.

01-35: ManA, a thermal tolerant mannanase to break down hemicellulose to sugars, which are then converted to ethanol via fermentation.

01-38: Gux 1, a thermostable cellulase with a GH48 catalytic domain, therefore potentially having exoglucanase activity.

01-36: AviIII, a thermostable cellulase with a GH74 catalytic domain.
BenefitsNREL’s enzyme improvements allow for economical conversion of biomass into biofuels. By increasing thermo stability, specific activity, and reducing inhibition, NREL’s enzyme portfolio can help your company to become competitive in the biofuels and biochemicals industry. Applications and Industries
  • Bio-fuels
  • Bio-polymers
  • Bio-chemicals
  • Pulp & paper
  • Detergents
  • Food & Feed processing
  • Textile processes
More InformationEndoglucanases
ROI: 89-05, Patent: 5,110,735
ROI: 95-56, Patent: 5,712,142
ROI: 94-08, Patent: 5,536,655
ROI: 93-92, Patent: 5,366,884
ROI: 89-05CIP, Patent: 5,275,944

Cellobiohydrolases
ROI: 03-05, Patent: 7,449,550
ROI: 99-45, Patent: 7,375,197

Beta-glucosidases
ROI: 93-21, Patent: 5,432,075

Dextranase
ROI: 98-37, Patent: 6,509,184

Mannanase
ROI: 01-35, Patent: 7,112,429

Gux 1
ROI: 01-38, Patent: 7,393,673

Avicelase
ROI: 01-36, Patent: 7,364,890

GuxA
ROI: 01-37, Patent: 7,059,993
Patents and Patent Applications
ID Number
Title and Abstract
Primary Lab
Date
Patent 5,110,735
Patent
5,110,735
Thermostable purified endoglucanase from thermophilic bacterium acidothermus cellulolyticus
A substantially purified high molecular weight cellulase enzyme having a molecular weight of between about 156,000 to about 203,400 daltons isolated from the bacterium Acidothermus cellulolyticus (ATCC 43068) and a method of producing it are disclosed. The enzyme is water soluble, possesses both C.sub.1 and C.sub.x types of enzymatic activity, has a high degree of stability toward heat and exhibits both a high optimum temperature activity and high inactivation characteristics.
National Renewable Energy Laboratory 05/05/1992
Issued
Patent 5,275,944
Patent
5,275,944
Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068
A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068). The cellulase is water soluble, possesses both C.sub.1 and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83.degree. C. at pH's from about 2 to about 9, and in inactivation temperature of about 110.degree. C. at pH's from about 2 to about 9.
National Renewable Energy Laboratory 01/04/1994
Issued
Patent 5,366,884
Patent
5,366,884
Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC
A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C.sub.1, and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81.degree. C. at pH's from about 2 to about 9, and at a inactivation temperature of about 100.degree. C. at pH's from about 2 to about 9.
National Renewable Energy Laboratory 11/22/1994
Issued
Patent 5,432,075
Patent
5,432,075
Low molecular weight thermostable .beta.-D-glucosidase from acidothermus cellulolyticus
A purified low molecular weight .beta.-D-glucosidase is produced from Acidothermus cellulolyticus ATCC 43068. The enzyme is water soluble, possesses activity against pNP-.beta.-D-glucopyranoside, has a high of degree of stability toward heat, exhibits optimal temperature activity at about 65.degree. C. at a pH range of from about 2 to about 7, has an inactivation temperature of about 80.degree. C. at a pH range of from about 2 to about 7 and has a molecular weight of about 50.5-54.5 kD as determineded by SDS-PAGE.
National Renewable Energy Laboratory 07/11/1995
Issued
Patent 5,536,655
Patent
5,536,655
Gene coding for the E1 endoglucanase
The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in heterologous microorganisms. A new modified E1 endoglucanase enzyme is produced along with variants of the gene and enzyme. The E1 endoglucanase is useful for hydrolyzing cellulose to sugars for simultaneous or later fermentation into alcohol.
National Renewable Energy Laboratory 07/16/1996
Issued
Patent 5,712,142
Patent
5,712,142
Method for increasing thermostability in cellulase ennzymes
The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in Pichia pastoris. A new modified E1 endoglucanase enzyme comprising the catalytic domain of the full size E1 enzyme demonstrates enhanced thermostability and is produced by two methods. The first method of producing the new modified E1 is proteolytic cleavage to remove the cellulose binding domain and linker peptide of the full size E1. The second method of producing the new modified E1 is genetic truncation of the gene encoding the full size E1 so that the catalytic domain is expressed in the expression product.
National Renewable Energy Laboratory 01/27/1998
Issued
Patent 6,509,184
Patent
6,509,184
Alkaline tolerant dextranase from streptomyces anulatus
A process for production of an alkaline tolerant dextranase enzyme comprises culturing a dextran-producing microorganism Streptomyces anulatus having accession no. ATCC PTA-3866 to produce an alkaline tolerant dextranase, Dex 1 wherein the protein in said enzyme is characterized by a MW of 63.3 kDa and Dex 2 wherein its protein is characterized by a MW of 81.8 kDa.
National Renewable Energy Laboratory 01/21/2003
Issued
Patent 7,059,993
Patent
7,059,993
Thermal tolerant cellulase from Acidothermus cellulolyticus
The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as GuxA. GuxA has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified GuxA. Methods of making and using GuxA polypeptides, including fusions, variants, and derivatives, are also disclosed.
National Renewable Energy Laboratory 06/13/2006
Issued
Patent 7,112,429
Patent
7,112,429
Thermal tolerant mannanase from acidothermus cellulolyticus
The invention provides a thermal tolerant mannanase that is a member of the glycoside hydrolase family. The invention further discloses this mannanase as ManA. ManA has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified ManA. Methods of making ManA polypeptides, including fusions, variants, and derivatives, are also disclosed. Methods of using mannanase A, including for the processing of food and for use in food stuffs as bulking agents and the like, are also disclosed.
National Renewable Energy Laboratory 09/26/2006
Issued
Patent 7,364,890
Patent
7,364,890
Thermal tolerant avicelase from Acidothermus cellulolyticus
The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from Acidothermus cellulolyticus and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.
National Renewable Energy Laboratory 04/29/2008
Issued
Patent 7,375,197
Patent
7,375,197
Cellobiohydrolase I gene and improved variants
The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.
National Renewable Energy Laboratory 05/20/2008
Issued
Patent 7,393,673
Patent
7,393,673
Thermal tolerant exoglucanase from Acidothermus cellulolyticus
The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as Gux1. Gux1 has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified Gux1. Methods of making and using Gux1 polypeptides, including fusions, variants, and derivatives, are also disclosed.
National Renewable Energy Laboratory 07/01/2008
Issued
Patent 7,449,550
Patent
7,449,550
Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum
Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.
National Renewable Energy Laboratory 11/11/2008
Issued
Technology Status
Development StageAvailabilityPublishedLast Updated
ProductionAvailable06/22/201007/28/2010

Contact NREL About This Technology

To: Eric Payne<Eric.Payne@nrel.gov>