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Compositions and methods for production of fermentable sugars

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20120270278 patent thumbnailZoom

Compositions and methods for production of fermentable sugars


The present application provides genetically modified fungal organisms that produce enzyme mixtures exhibiting enhanced hydrolysis of cellulosic material to glucose, enzyme mixtures produced by the genetically modified fungal organisms, and processes for producing glucose from cellulose using such enzyme mixtures.

Browse recent Codexis, Inc. patents - Redwood City, CA, US
Inventors: ISH KUMAR DHAWAN, DIPNATH BAIDYAROY, ANDREW SHAW, OLEH TANCHAK, CHRISTOPHER HILL, CHENGSONG LIU, AMALA CHOKSHI, BRIAN R. SCOTT
USPTO Applicaton #: #20120270278 - Class: 435100 (USPTO) - 10/25/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition >Preparing Compound Containing Saccharide Radical >Disaccharide



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The Patent Description & Claims data below is from USPTO Patent Application 20120270278, Compositions and methods for production of fermentable sugars.

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The present application is a Divisional of U.S. patent application Ser. No. 13/286,972, filed Nov. 1, 2011, which claims priority to U.S. Prov. Patent Appln. Ser. Nos. 61/409,186, 61/409,217, 61/409,472, and 61/409,480, all of which were filed on Nov. 2, 2010, and are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention provides compositions and methods for the production of fermentable sugars. In some embodiments, the present invention provides genetically modified fungal organisms. In some additional embodiments, the present invention provides enzymes that find use in enhancing hydrolysis of cellulosic material to fermtable sugars (e.g., glucose), and methods for using the enzymes. In some further embodiments, the present invention provides enzyme mixtures useful for the hydrolysis of cellulosic materials.

BACKGROUND

Cellulose is a polymer of the simple sugar glucose linked by beta-1,4 glycosidic bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and lower overall greenhouse gas production. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the cellulose is converted to glucose, the glucose is easily fermented by yeast into ethanol.

Although progress has been made in increasing the efficiency of enzymatic degradation of lignocellulosic feedstocks, there remains a great need to improve yield of fermentable sugars using enzymatic processes.

SUMMARY

OF THE INVENTION

The present invention provides genetically modified fungal organisms, as well as enzymes that enhance hydrolysis of cellulosic material to glucose, and methods for using the enzymes.

The present invention provides fungal cells that have been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is produced by the fungal cells. In some embodiments, the fungal cell is an Ascomycete belonging to the subdivision Pezizomycotina, and/or wherein the fungal cell is from the family Chaetomiaceae. In some embodiments, the fungal cell is a species of Myceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, Corynascus, Acremonium, Ctenomyces, Chrysosporium, Scytalidium, Talaromyces, Thermoascus, or Aspergillus. In some additional embodiments, the fungal cell is a species of Myceliophthora, Thielavia, Sporotrichum, Chrysosporium, Corynascus, Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus, while in some other embodiments, the fungal cell is Sporotrichum thermophile Sporotrichum cellulophilum, Thielavia heterothallica, Thielavia terrestris, Corynascus heterothallicus, or Myceliophthora thermophile. In some embodiments, the fungal cell has been genetically modified to reduce the amount of endogenous glucose oxidase and/or cellobiose dehydrogenase that is produced by the fungal cell. In some additional embodiments, the fungal cell has been genetically modified to reduce the amount of endogenous glucose oxidase and/or cellobiose dehydrogenase that is produced by the fungal cell and to increase the production of at least one saccharide hydrolyzing enzyme. In some further embodiments, the fungal cell has been genetically modified to reduce the amount of endogenous glucose oxidase and/or cellobiose dehydrogenase that is produced by the fungal cell and to increase the production of at least one saccharide hydrolyzing enzyme, and wherein the fungal cell is a Basidiomycete belonging to the class Agaricomycetes. In some embodiments, the Basidiomycete is a species of Pleurotus, Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex. In some embodiments, the fungal cell has been genetically modified to reduce the amount of the endogenous glucose oxidase and/or cellobiose dehydrogenase that is secreted by the fungal cell. In some additional embodiments, the fungal cell has been genetically modified to disrupt the secretion signal peptide of the glucose and/or cellobiose oxidizing enzyme. In some further embodiments, the fungal cell has been genetically modified to reduce the amount of the endogenous glucose and/or cellobiose oxidizing enzyme that is expressed by the fungal cell. In still some additional embodiments, the fungal cell has been genetically modified to disrupt a translation initiation sequence in the transcript encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In some additional embodiments, the fungal cell has been genetically modified to introduce a frameshift mutation in the transcript encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In some further embodiments, the fungal cell has been genetically modified to reduce the transcription level of a gene encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In some embodiments, the fungal cell has been genetically modified to disrupt the promoter of a gene encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In still some additional embodiments, the fungal cell has been genetically modified to at least partially delete at least one gene encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In some further embodiments, the fungal cell has been genetically modified to reduce the catalytic efficiency of the endogenous glucose and/or cellobiose oxidizing enzyme. In some additional embodiments, the fungal cell has been genetically modified to mutate one or more residues in an active site of the glucose and/or cellobiose oxidizing enzyme. In some further embodiments, the fungal cell has been genetically modified to mutate one or more residues in a heme binding domain of the glucose and/or cellobiose oxidizing enzyme. In some embodiments of the fungal cells provided herein, the glucose and/or cellobiose oxidizing enzyme is selected from cellobiose dehydrogenase (EC 1.1.99.18), glucose oxidase (EC 1.1.3.4), pyranose oxidase (EC1.1.3.10), glucooligosaccharide oxidase (EC 1.1.99.B3), pyranose dehydrogenase (EC 1.1.99.29), and glucose dehydrogenase (EC 1.1.99.10). In some additional embodiments, the glucose and/or cellobiose oxidizing enzyme comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, and/or 16. In some further embodiments, the glucose and/or cellobiose oxidizing enzyme is cellobiose dehydrogenase (EC 1.1.99.18). In some embodiments, the fungal cell has been genetically modified to reduce the amount of glucose and/or cellobiose oxidizing enzyme activity of two or more endogenous glucose and/or cellobiose oxidizing enzymes that are produced by the fungal cell prior to genetic modification. In some further embodiments, the first of the two or more the glucose and/or cellobiose oxidizing enzymes comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and/or 16, and a second of the two or more the glucose and/or cellobiose oxidizing enzymes comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and/or 16.

The present invention also provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is expressed by at least one of the fungal cells provided herein.

The present invention also provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell, and wherein the fungal cell is an Ascomycete belonging to the subdivision Pezizomycotina. In some embodiments, the fungal cell is a species of Myceliophthora, Thielavia, Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus.

The present invention also provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell and to increase the production of at least one saccharide hydrolyzing enzyme, wherein the fungal cell is a Basidiomycete belonging to the class Agaricomycetes.

In some embodiments, the enzyme mixtures are cell-free mixtures. In some additional embodiments, a substrate of the enzyme mixture comprises pretreated lignocellulose. In some further embodiments, the pretreated lignocellulose comprises lignocellulose treated by a treatment method selected from acid pretreatment, ammonium pretreatment, steam explosion and/or organic solvent extraction.

The present invention also provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, wherein the fungal cellulase enzyme mixture is modified relative to a parental (or reference) enzyme mixture to be at least partially deficient in glucose and/or cellobiose oxidizing enzyme activity.

The present invention further provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, at least one of the cellulose hydrolyzing enzymes being endogenous to a fungal cell, wherein the fungal cell is a Basidiomycete belonging to the class Agaricomycetes or an Ascomycete belonging to the subdivision Pezizomycotina and wherein the enzyme mixture is characterized in that, when the enzyme mixture is contacted with cellobiose and/or glucose, no more than about 10%, about 15% or about 20%, of the cellobiose and/or glucose is oxidized after 10 hours.

In some embodiments of the enzyme mixtures, the fungal cell has been genetically modified to reduce the amount of glucose and/or cellobiose oxidase enzyme activity that is secreted by the fungal cell. In some further embodiments, the enzyme mixture is a cell-free mixture. In some additional embodiments, the enzyme mixture comprises at least one beta-glucosidase. In some further embodiments, the enzyme mixture comprises at least one cellulase enzyme selected from endoglucanases (EGs), beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s), and/or variants of the cellulase enzyme. In some embodiments, the enzyme mixture further comprises at least one cellobiose dehydrogenase. In some embodiments, the celliobiose dehydrogenase is CDH1 and/or CDH2. In some additional embodiments, the enzyme mixture further comprises at least one cellulase enzyme and/or at least one additional enzyme. In some further embodiments, the enzyme mixture has been subjected to a purification process to selectively remove one or more glucose and/or cellobiose oxidizing enzymes from the enzyme mixture. In some embodiments, the purification process comprises selective precipitation to separate the glucose and/or cellobiose oxidizing enzymes from other enzymes present in the enzyme mixture. In some additional embodiments, the enzyme mixtures comprise at least one inhibitor of one or more glucose and/or cellobiose oxidizing enzymes.

The present invention also provides methods for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell that is an Ascomycete belonging to the subdivision Pezizomycotina, and wherein the enzyme mixture is characterized in that, when the enzyme mixture is contacted with cellobiose and/or glucose, no more than about 10%, about 15%, or about 20% of the cellobiose and/or glucose is oxidized after 10 hours. In some embodiments, the Ascomycete is a species of Myceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, or Aspergillus.

The present invention also provides methods for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell that is a Basidiomycete belonging to the class Agaricomycetes, and wherein the enzyme mixture is characterized in that, when the enzyme mixture is contacted with cellobiose and/or glucose, no more than about 10%, about 15% or about 20% of the cellobiose and/or glucose is oxidized after 10 hours. In some embodiments, the Basidiomycete is a species of Pleurotus, Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.

The present invention also provides methods for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell that is an Ascomycete belonging to the subdivision Pezizomycotina, and wherein, of the cellulose hydrolyzed by the enzyme mixture, at least about 80%, about 85%, or about 90% is present in the form of cellobiose and/or glucose. In some embodiments, the Ascomycete is a species of Myceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, or Aspergillus. In some embodiments, the Ascomycete is Myceliophthora thermophila, Thielavia heterothallica or Sporotrichum thermophile. In some embodiments, the fungal cell is Myceliophthora thermophila.

The present invention also provides methods for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell that is a Basidiomycete belonging to the class Agaricomycetes, and wherein, of the cellulose hydrolyzed by the enzyme mixture, at least about 80%, about 85%, or about 90% is present in the form of cellobiose and/or glucose. In some embodiments, the Basidiomycete is a species of Pleurotus, Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.

The present invention also provides methods for producing cellobiose and/or glucose from cellulose comprising treating a cellulose substrate with an enzyme mixture to generate glucose, wherein the enzyme mixture is modified relative to a secreted enzyme mixture from a reference (or parental) fungal cell to be at least partially deficient in glucose and/or cellobiose oxidizing enzyme activity. In some embodiments of the methods, the enzyme mixture is a cell-free mixture. In some additional embodiments, the cellulose substrate comprises pretreated lignocellulose. In some further embodiments, the pretreated lignocellulose comprises lignocellulose treated by a treatment method selected from acid pretreatment, ammonium pretreatment, steam explosion and/or organic solvent extraction. In some further embodiments, the methods further comprise fermentation of the cellobiose and/or glucose to at least one end product. In some embodiments, the end product is at least one fuel alcohol and/or at least one precursor industrial chemical. In some additional embodiments, the fuel alcohol is ethanol or butanol. In some embodiments, the process for producing cellobiose and/or glucose from cellulose and said fermentation are conducted in a simultaneous saccharification and fermentation (SSF) process. In some further additional embodiments, the enzyme mixture is produced by a fungal cell has that been genetically modified to reduce the amount of one or more endogenous glucose and/or cellobiose oxidizing enzymes that is secreted by the fungal cell. In some embodiments, the enzyme mixture has been subjected to a purification process to selectively remove at least one glucose and/or cellobiose oxidizing enzyme from the enzyme mixture. In some further embodiments, the purification process comprises selective precipitation to separate the glucose and/or cellobiose oxidizing enzyme from other enzymes present in the enzyme mixture. In still some additional embodiments, the enzyme mixture comprises at least one inhibitor of the glucose and/or cellobiose oxidizing enzyme. In some embodiments, the inhibitor comprises a broad-spectrum oxidase inhibitor selected from sodium azide, potassium cyanide, a metal anion, and a combination thereof. In some embodiments, the inhibitor comprises a specific inhibitor of cellobiose dehydrogenase (EC 1.1.99.18) selected from cellobioimidazole, gentiobiose, lactobiono-1,5-lactone, celliobono-1,5-lactone, tri-N-acetylchitortriose, methyl-beta-D cellobiosidase, 2,2-bipyridine, cytochrome C, and a combination thereof. In some embodiments, the method is a batch process, while in some other embodiments it is a continuous process, and in some further embodiments it is a fed-batch process and in still further embodiments, it is a combination of batch, continuous and/or fed-batch processes conducted in any order. In some embodiments, the method is conducted in a reaction volume of at least 10,000 liters, while in some other embodiments, the method is conducted in a reaction volume of at least 100,000 liters. In some embodiments, the enzyme mixture comprises at least one beta-glucosidase, while in some other embodiments, the enzyme mixture does not comprise a beta-glucosidase. In some embodiments, the enzyme mixture comprises at least one endoglucanase, while in some other embodiments, the enzyme mixture does not comprise an endoglucanase. In some embodiments, the enzyme mixture comprises at least one cellulase enzyme selected from endoglucanases (EGs), beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s), and/or variants of said cellulase enzyme.

The present invention also provides methods for generating glucose comprising contacting cellulose with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by the fungal cells provided herein.

The present invention also provides methods for generating glucose comprising contacting cellulose with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell, wherein the fungal cell is an Ascomycete belonging to the subdivision Pezizomycotina. In some embodiments, the fungal cell is a species of Myceliophthora, Thielavia, Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus.

The present invention also provides methods for generating glucose comprising contacting cellulose with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell and to increase the production of at least one saccharide hydrolyzing enzyme, wherein the fungal cell is a Basidiomycete belonging to the class Agaricomycetes.

The present invention further provides methods for generating glucose comprising contacting cellulose with at least one enzyme mixture as provided herein. In some embodiments, the cellulose comprises pretreated lignocellulose. In some additional embodiments, the pretreated lignocellulose comprises lignocellulose treated by a treatment method selected from acid pretreatment, ammonium pretreatment, steam explosion and/or organic solvent extraction. In some additional embodiments, the enzyme mixture is a cell-free mixture. In some further embodiments, the methods further comprise fermentation of the glucose to an end product. In some embodiments, the end product is a fuel alcohol or a precursor industrial chemical. In some embodiments, the fuel alcohol is ethanol or butanol.

The present invention further provides the fungal cells provided herein, as well s the enzyme mixtures provided herein, and the methods provided herein, further comprising a cellulose degrading enzyme that is heterologous to the fungal cell.

The present invention also provides fermentation media comprising at least one fungal cell provided herein.

The present invention also provides fermentation media comprising at least one enzyme mixture provided herein.

The present invention further provides fermentation media comprising at least one fungal cell and/or at least one enzyme mixture, as provided herein.

The present invention also provides methods of producing at least one cellulase, comprising at least one fungal cell provided herein, under conditions such that said at least one cellulase is produced. In some embodiments, the fungal cell is recombinant.

The present invention also provides compositions comprising at least one cellulase as provided herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart that shows the products of cellulose hydrolysis using enzyme mixtures obtained from strains CF-402, CF-403, and CF-401 as further described in Example 1 and Example 7. Dark bars represent measured glucose production. Light bars represent measured gluconate production. Numbers above horizontal bars indicate the sum of glucose and gluconate fractions.

FIG. 2 is a chart that shows the products of cellulose hydrolysis using enzyme mixtures produced by strain CF-400 (comprising a cdh1 deletion); strain CF-401 (comprising the deletions of cdh1 and cdh2) and strain CF-402 (comprising cdh1 and cdh2), as further described in Example 8.

FIGS. 3 and 4 provide the nucleotide and amino acid sequences of M. thermophila CDH1 and CDH2 (SEQ ID NOS:5-8).

FIGS. 5 and 6 provide the nucleotide and amino acid sequences of M. thermophila GO1 and GO2 (SEQ ID NOS:1-4).

FIG. 7 provides the nucleotide and amino acid sequences of A. oryzae pyranose oxidase (SEQ ID NOS:9-10).

FIG. 8 provides the nucleotide and amino acid sequences of A. strictum glucooligosaccharide oxidase (SEQ ID NOS:11-12).

FIG. 9 provides the nucleotide and amino acid sequences of A. bisporus pyranose dehydrogenase (SEQ ID NOS:13-14).

FIG. 10 provides the nucleotide and amino acid sequences of T. stipitatus ATCC10500 glucose dehydrogenase (SEQ ID NOS:15-16).

FIG. 11 provides a chart showing fractional recovery of available cellulose using an enzyme mixture containing cellobiose dehydrogenase activity. Dark bars represent glucose yield as measured using a horseradish peroxidase coupled enzymatic assay described in Example 1. Light bars represent expected glucose yield calculated using the IR method for determining cellulose conversion described in Example 9.

FIGS. 12A and 12B are HPLC chromatograms showing the effect of acid hydrolysis of cellotriose (FIG. 12A) or of cellulose hydrolysis products produced by an enzyme mixture containing cellobiose dehydrogenase (FIG. 12B) as described in Example 11.

FIG. 13 provides an IR spectrum of cellulose hydrolysate obtained using enzyme mixtures lacking (Turbo) or containing (CF-402) cellobiose dehydrogenase activity. The vertical arrow indicates the carbonyl peak at 1715 cm−1 unique to the hydrolysate produced by the CF-402 enzyme mixture.

FIGS. 14A and 14B are HPLC chromatograms that identify an oxidized glucose product produced from glucose (FIG. 5A) or from cellulose hydrolysate using cellulase enzymes secreted by strain CF-402, as described in Example 13.

DESCRIPTION OF THE INVENTION

The present invention provides genetically modified fungal organisms, as well as enzymes that enhance hydrolysis of cellulosic material to glucose, and methods for using the enzymes.

All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference. Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, fermentation, microbiology, and related fields, which are known to those of skill in the art. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some suitable methods and materials are described. Indeed, it is intended that the present invention not be limited to the particular methodology, protocols, and reagents described herein, as these may vary, depending upon the context in which they are used. The headings provided herein are not limitations of the various aspects or embodiments of the present invention.

Nonetheless, in order to facilitate understanding of the present invention, a number of terms are defined below. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “host cell” includes a plurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of the invention that can be had by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

As used herein, “substrate” refers to a substance or compound that is converted or meant to be converted into another compound by the action of an enzyme. The term includes not only a single compound but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate.

As used herein, “conversion” refers to the enzymatic transformation of a substrate to the corresponding product. “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, for example, the “enzymatic activity” or “activity” of a cellobiose dehydrogenase (“CDH” or “cdh”) polypeptide can be expressed as “percent conversion” of the substrate to the product.

As used herein, “secreted activity” refers to enzymatic activity of glucose and/or cellobiose oxidizing enzymes produced by a fungal cell that is present in an extracellular environment. An extracellular environment can be, for example, an extracellular milieu such as a culture medium. The secreted activity is influenced by the total amount of glucose and/or cellobiose oxidizing enzyme secreted, and also is influenced by the catalytic efficiency of the secreted glucose and/or cellobiose oxidizing enzyme.

As used herein, a “reduction in catalytic efficiency” refers to a reduction in the activity of the glucose and/or cellobiose oxidizing enzyme, relative to unmodified glucose and/or cellobiose oxidizing enzyme, as measured using standard techniques, as provided herein or otherwise known in the art.

As used herein, the term “enzyme mixture” refers to a combination of at least two enzymes. In some embodiments, at least two enzymes are present in a composition. In some additional embodiments, the enzyme mixtures are present within a cell (e.g., a fungal cell). In some embodiments, each or some of the enzymes present in an enzyme mixture are produced by different fungal cells and/or different fungal cultures. In some further embodiments, all of the enzymes present in an enzyme mixture are produced by the same cell. In some embodiments, the enzyme mixtures comprise cellulase enzymes, while in some additional embodiments, the enzyme mixtures comprise enzymes other than cellulases. In some embodiments, the enzyme mixtures comprise at least one cellulase and at least one enzyme other than a cellulase. In some embodiments, the enzyme mixtures comprise enzymes including, but not limited to endoxylanases (EC 3.2.1.8), beta-xylosidases (EC 3.2.1.37), alpha-L-arabinofuranosidases (EC 3.2.1.55), alpha-glucuronidases (EC 3.2.1.139), acetylxylanesterases (EC 3.1.1.72), feruloyl esterases (EC 3.1.1.73), coumaroyl esterases (EC 3.1.1.73), alpha-galactosidases (EC 3.2.1.22), beta-galactosidases (EC 3.2.1.23), beta-mannanases (EC 3.2.1.78), beta-mannosidases (EC 3.2.1.25), endo-polygalacturonases (EC 3.2.1.15), pectin methyl esterases (EC 3.1.1.11), endo-galactanases (EC 3.2.1.89), pectin acetyl esterases (EC 3.1.1.6), endo-pectin lyases (EC 4.2.2.10), pectate lyases (EC 4.2.2.2), alpha rhamnosidases (EC 3.2.1.40), exo-galacturonases (EC 3.2.1.82), exo-galacturonases (EC 3.2.1.67), exopolygalacturonate lyases (EC 4.2.2.9), rhamnogalacturonan endolyases EC (4.2.2.B3), rhamnogalacturonan acetylesterases (EC 3.2.1.B11), rhamnogalacturonan galacturonohydrolases (EC 3.2.1.B11), endo-arabinanases (EC 3.2.1.99), laccases (EC 1.10.3.2), manganese-dependent peroxidases (EC 1.10.3.2), amylases (EC 3.2.1.1), glucoamylases (EC 3.2.1.3), lipases, lignin peroxidases (EC 1.11.1.14), and/or proteases.

In some additional embodiments, the present invention further provides enzyme mixtures comprising at least one expansin and/or expansin-like protein, such as a swollenin (See e.g., Salheimo et al., Eur. J. Biochem., 269:4202-4211 [2002]) and/or a swollenin-like protein. Expansins are implicated in loosening of the cell wall structure during plant cell growth. Expansins have been proposed to disrupt hydrogen bonding between cellulose and other cell wall polysaccharides without having hydrolytic activity. In this way, they are thought to allow the sliding of cellulose fibers and enlargement of the cell wall. Swollenin, an expansin-like protein contains an N-terminal Carbohydrate Binding Module Family 1 domain (CBD) and a C-terminal expansin-like domain. In some embodiments, an expansin-like protein and/or swollenin-like protein comprises one or both of such domains and/or disrupts the structure of cell walls (e.g., disrupting cellulose structure), optionally without producing detectable amounts of reducing sugars. In some additional embodiments, the enzyme mixtures comprise at least one polypeptide product of a cellulose integrating protein, scaffoldin and/or a scaffoldin-like protein (e.g., CipA or CipC from Clostridium thermocellum or Clostridium cellulolyticum respectively). In some additional embodiments, the enzyme mixtures comprise at least one cellulose induced protein and/or modulating protein (e.g., as encoded by cip1 or cip2 gene and/or similar genes from Trichoderma reesei; See e.g., Foreman et al., J. Biol. Chem., 278:31988-31997 [2003]). In some additional embodiments, the enzyme mixtures comprise at least one member of each of the classes of the polypeptides described above, several members of one polypeptide class, or any combination of these polypeptide classes to provide enzyme mixtures suitable for various uses.

Any combination of at least one two, three, four, five, or more than five enzymes and/or polypeptides find use in various enzyme mixtures provided herein. Indeed, it is not intended that the enzyme mixtures of the present invention be limited to any particular enzymes, polypeptides, nor combinations, as any suitable enzyme mixture finds use in the present invention.

As used herein, the term “saccharide” refers to any carbohydrate comprising monosaccharides (e.g., glucose, ribose, fructose, galactose, etc.), disaccharides (e.g., sucrose, lactose, maltose, cellobiose, trehalose, melibiose, etc.), oligosaccharides (e.g., raffinose, stachyose, amylose, etc.), and polysaccharides (e.g., starch, glycogen, cellulose, chitin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, callose, laminarin, chrysolaminarin, amylopectin, dextran, dextrins, maltodextrins, inulin, oligofructose, polydextrose, etc.). The term encompasses simple carbohydrates, as well as complex carbohydrates. Indeed, it is not intended that the present invention be limited to any particular saccharide, as various saccharides and forms of saccharides find use in the present invention.

As used herein, the term “saccharide hydrolyzing enzyme” refers to any enzyme that hydrolyzes at least one sachharide.

As used herein, the terms “glucose oxidizing enzyme” and “cellobiose oxidizing enzyme” refer to enzymes that oxidize glucose and/or cellobiose. For example, glucose and/or cellobiose oxidizing enzymes include glucose oxidase (EC 1.1.3.4), cellobiose dehydrogenase (EC 1.1.99.18), pyranose oxidase (EC 1.1.3.10), glucooligosaccharide oxidase (EC 1.1.99.B3), pyranose dehydrogenase (EC 1.1.99.29), and glucose dehydrogenase (EC 1.1.99.10).

As used herein, the terms “glucose oxidase” and “GO” refer to an enzyme that is an oxido-reductase that catalyses the oxidation of β-D-glucose into D-glucono-1,5-lactone, which is a cyclic ester existing at a pH-dependent equilibrium in aqueous solution with gluconic acid or gluconate. Exemplary glucose oxidases fall into the enzyme classification (EC 1.1.3.4). In order to work as a catalyst, glucose oxidases typically utilize a co-substrate oxidant, such as flavin adenine dinucleotide (FAD). The enzyme is highly specific for β-D-glucose. However, glucose oxidase also can demonstrate some lesser oxidase activity for substrates 2-deoxy-D-glucose, D-mannose and D-galactose (See e.g., Bentley, Meth. Enzymol., 9:86 [1996]).

As useded herein, the terms “cellobiose dehydrogenase” and “CDH” refer to a cellobiose:acceptor 1-oxidoreductase that catalyzes the conversion of cellobiose in the presence of an acceptor to cellobiono-1,5-lactone and a reduced acceptor. Examples of cellobiose dehydrogenases fall into the enzyme classification (E.C. 1.1.99.18). Typically 2,6-Dichloroindophenol can act as acceptor, as can iron, especially Fe(SCN)3, molecular oxygen, ubiquinone, or cytochrome C, and other polyphenolics, such as lignin. Substrates of the enzyme include cellobiose, cello-oligosaccharides, lactose, and D-glucosyl-1,4-β-D-mannose, glucose, maltose, mannobiose, thiocellobiose, galactosyl-mannose, xylobiose, xylose. Electron donors include beta-1-4 dihexoses with glucose or mannose at the reducing end, though alpha-1-4 hexosides, hexoses, pentoses, and beta-1-4 pentomers can act as substrates for at least some of these enzymes (See e.g., Henriksson et al, Biochim. Biophys. Acta-Prot. Struct. Mol. Enzymol., 1383: 48-54 [1998]; and Schou et al., Biochem. J., 330: 565-571 [1998]).

As used herein, the terms “oxidation”, “oxidize(d)” and the like as used herein refer to the enzymatic formation of one or more glucose or cellobiose oxidation products including, but not limited to, cellobionolactone, cellobionic acid, gluconolactone, gluconate and/or gluconic acid. When used in reference to a percentage of oxidized cellobiose and/or glucose, those percentages reflect a weight percent (w/w) relative to the initial amount of substrate. For example, when the enzyme mixture is contacted with cellobiose and/or glucose, the percentage of oxidized cellobiose and/or glucose reflects a weight percent (w/w) relative to the initial amount of cellobiose and/or glucose present in solution. Where the enzyme mixture is contacted with a cellulose substrate, the percentage of oxidized cellobiose and/or glucose reflects a weight percent (w/w) based on the maximum amount (wt %) of glucose that could be produced from the total hydrolyzed cellulose (i.e., Gmax).

As used herein, the terms “cellobiose dehydrogenase” and “CDH” refer to a cellobiose:acceptor 1-oxidoreductase that catalyzes the conversion of cellobiose in the presence of an acceptor to cellobiono-1,5-lactone and a reduced acceptor. Examples of cellobiose dehydrogenases are included in the enzyme classification (E.C. 1.1.99.18). In some embodiments, the cellobiose dehydrogenase of interest in the present invention is CDH1, which is encoded by the cdh1 gene. In some embodiments, the cellobiose dehydrogenase of interest in the present invention is CDH2, which is encoded by the cdh2 gene. In some embodiments, both CDH1 and CDH2 are of interest.

As used herein, the terms “pyranose oxidase” and “PO” refer to an enzyme that catalyzes the conversion of D-glucose and O2 to 2-dehydro-D-glucose and H2O2. Examples of pyranose oxidases fall into the enzyme classification (E.C. 1.1.3.10). The systematic name of this enzyme class is pyranose:oxygen 2-oxidoreductase. Other names in common use include glucose 2-oxidase, and pyranose-2-oxidase. Substrates of the enzyme include D-glucose, D-xylose, L-arabinose, L-sorbose, D-glucono-1,5-lactone, cellobiose and gentiobiose.

As used herein, the terms “glucooligosaccharide oxidase” and “GOOX” refer to an enzyme that catalyzes the oxidation of oligosaccharides with glucose on the reducing end and each sugar residue joined by an alpha- or beta-1,4 glucosidic bond. Examples of glucooligosaccharide oxidase fall into the enzyme classification (E.C. 1.1.99.B3). The systematic name of this enzyme class is carbohydrate:acceptor oxidoreductase. Substrates of the enzyme include maltose, lactose, cellobiose and maltose derivatives up to seven residues.

As used herein, the terms “pyranose dehydrogenase” and “PDH” refer to an enzyme that catalyzes the reaction of pyranose and an acceptor to yield 2-dehydropyranose (or 3-dehydropyranose or 2,3-didehydropyranose) and a reduced acceptor. PDH also catalyzes the reaction of a pyranoside and an acceptor to yield a 3-dehydropyranoside (or 3,4-didehydropyranoside) and a reduced acceptor. Examples of pyranose dehydrogenases fall into the enzyme classification (E.C. 1.1.99.29). The systematic name of this enzyme class is pyranose:acceptor oxidoreductase. Other names in common use include pyranose 2,3-dehydrogenase. PDH utilizes FAD as a cofactor. A number of aldoses and ketoses in pyranose form, as well as glycosides, gluco-oligosaccharides, sucrose and lactose can act as a donor. 1,4-Benzoquinone or ferricenium ion (ferrocene oxidized by removal of one electron) can serve as acceptor. Unlike EC 1.1.3.10 (pyranose oxidase), pyranose dehydrogenase does not interact with O2 and exhibits extremely broad substrate tolerance with variable regioselectivity (C-3, C-2 or C-3+C-2 or C-3+C-4) for (di)oxidation of different sugars. D-Glucose is exclusively or preferentially oxidized at C-3 (depending on the enzyme source), but can also be oxidized at C-2+C-3. Pyranose dehydrogenase also acts on 1->4-alpha- and 1->4-beta-gluco-oligosaccharides, non-reducing gluco-oligosaccharides and L-arabinose, which are not substrates of EC 1.1.3.10. Sugars are oxidized by pyranose dehydrogenase in their pyranose but not in their furanose form.

As used herein, the terms “glucose dehydrogenase” and “GDH” refer to an enzyme that catalyzes the reaction of D-glucose and an acceptor to yield D-glucono-1,5-lactone and a reduced acceptor. Examples of glucose dehydrogenase fall into the enzyme classification (E.C. 1.1.99.10). The systematic name of this enzyme class is D-glucose:acceptor 1-oxidoreductase. GDH utilizes FAD as a cofactor.

As used herein, the term “cellulase” refers to any enzyme that is capable of degrading cellulose. Thus, the term encompasses enzymes capable of hydrolyzing cellulose (β-1,4-glucan or β-D-glucosidic linkages) to shorter cellulose chains, oligosaccharides, cellobiose and/or glucose. “Cellulases” are divided into three sub-categories of enzymes: 1,4-β-D-glucan glucanohydrolase (“endoglucanase” or “EG”); 1,4-β-D-glucan cellobiohydrolase (“exoglucanase,” “cellobiohydrolase,” or “CBH”); and β-D-glucoside-glucohydrolase (“β-glucosidase,” “cellobiase,” “BG,” or “BGL”). These enzymes act in concert to catalyze the hydrolysis of cellulose-containing substrates. Endoglucanases break internal bonds and disrupt the crystalline structure of cellulose, exposing individual cellulose polysaccharide chains (“glucans”). Cellobiohydrolases incrementally shorten the glucan molecules, releasing mainly cellobiose units (a water-soluble β-1,4-linked dimer of glucose) as well as glucose, cellotriose, and cellotetrose. Beta-glucosidases split the cellobiose into glucose monomers.

Cellulases often comprise a mixture of different types of cellulolytic enzymes (endoglucanases and cellobiohydrolases) that act synergistically to break down the cellulose to soluble di- or oligosaccharides such as cellobiose, which are then further hydrolyzed to glucose by beta-glucosidase. Cellulase enzymes are produced by a wide variety of microorganisms. Cellulases (and hemicellulases) from filamentous fungi and some bacteria are widely exploited for many industrial applications that involve processing of natural fibers to sugars.

As used herein, a “cellulase-producing fungal cell” is a fungal cell that produces at least one cellulase enzyme (i.e., “cellulose hydrolyzing enzyme”). In some embodiments, the cellulase-producing fungal cells provided herein express and secrete a mixture of cellulose hydrolyzing enzymes. As used herein, the terms “cellulose hydrolyzing enzyme,” “cellulolytic enzyme,” and like terms refer to an enzyme that acts in the process of breaking down cellulose to soluble di- or oligosaccharides such as cellobiose, which are then further hydrolyzed to glucose by beta-glucosidase. A mixture of cellulose hydrolyzing enzymes is also referred to herein as “cellulases,” a “cellulase-containing mixture,” and/or a “cellulase mixture.”

As used herein, the terms “endoglucanase” and “EG” refer to a category of cellulases (EC 3.2.1.4) that catalyze the hydrolysis of internal β-1,4 glucosidic bonds of cellulose. The term “endoglucanase” is further defined herein as an endo-1,4-(1,3; 1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4), which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenan, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined based on a reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (See e.g., Zhang et al., Biotechnol. Adv., 24:452-481 [2006]). For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) hydrolysis (See e.g., Ghose, Pur. Appl. Chem., 59:257-268 [1987]).

As used herein, “EG1” refers to a carbohydrate active enzyme expressed from a nucleic acid sequence coding for a glycohydrolase (GH) Family 7 catalytic domain classified under EC 3.2.1.4 or any protein, polypeptide or catalytically active fragment thereof. In some embodiments, the EG1 is functionally linked to a carbohydrate binding module (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG2” refers to a carbohydrate active enzyme expressed from a nucleic acid sequence coding for a glycohydrolase (GH) Family 5 catalytic domain classified under EC 3.2.1.4 or any protein, polypeptide or catalytically active fragment thereof. In some embodiments, the EG2 is functionally linked to a carbohydrate binding module (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG3” refers to a carbohydrate active enzyme expressed from a nucleic acid sequence coding for a glycohydrolase (GH) Family 12 catalytic domain classified under EC 3.2.1.4 or any protein, polypeptide or catalytically active fragment thereof. In some embodiments, the EG3 is functionally linked to a carbohydrate binding module (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG4” refers to a carbohydrate active enzyme expressed from a nucleic acid sequence coding for a glycohydrolase (GH) Family 61 catalytic domain classified under EC 3.2.1.4 or any protein, polypeptide or fragment thereof. In some embodiments, the EG4 is functionally linked to a carbohydrate binding module (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG5” refers to a carbohydrate active enzyme expressed from a nucleic acid sequence coding for a glycohydrolase (GH) Family 45 catalytic domain classified under EC 3.2.1.4 or any protein, polypeptide or fragment thereof. In some embodiments, the EG5 is functionally linked to a carbohydrate binding module (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG6” refers to a carbohydrate active enzyme expressed from a nucleic acid sequence coding for a glycohydrolase (GH) Family 6 catalytic domain classified under EC 3.2.1.4 or any protein, polypeptide or fragment thereof. In some embodiments, the EG6 is functionally linked to a carbohydrate binding module (CBM), such as a Family 1 cellulose binding domain.

As used herein, the terms “cellobiohydrolase” and “CBH” refer to a category of cellulases (EC 3.2.1.91) that hydrolyze glycosidic bonds in cellulose. The term “cellobiohydrolase” is further defined herein as a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain (See e.g., Teeri, Tr. Biotechnol., 15:160-167 [1997]; and Teeri et al., Biochem. Soc. Trans., 26:173-178 [1998]). In some embodiments, cellobiohydrolase activity is determined using a fluorescent disaccharide derivative 4-methylumbelliferyl-.beta.-D-lactoside (See e.g., van Tilbeurgh et al., FEBS Lett., 149:152-156 [1982]; and van Tilbeurgh and Claeyssens, FEBS Lett., 187:283-288 [1985]).

As used herein, the terms “CBH1” and “type 1 cellobiohydrolase” refer to a carbohydrate active enzyme expressed from a nucleic acid sequence coding for a glycohydrolase (GH) Family 7 catalytic domain classified under EC 3.2.1.91 or any protein, polypeptide or catalytically active fragment thereof. In some embodiments, the CBH1 is functionally linked to a carbohydrate binding module (CBM), such as a Family 1 cellulose binding domain.

As used herein, the terms “CBH2” and “type 2 cellobiohydrolase” refer to a carbohydrate active enzyme expressed from a nucleic sequence coding for a glycohydrolase (GH) Family 6 catalytic domain classified under EC 3.2.1.91 or any protein, polypeptide or catalytically active fragment thereof. Type 2 cellobiohydrolases are also commonly referred to as “the Cel6 family.” In some embodiments, the CBH2 is functionally linked to a carbohydrate binding module (CBM), such as a Family 1 cellulose binding domain.

As used herein, the terms “beta-glucosidase,” “cellobiase,” and “BGL” refers to a category of cellulases (EC 3.2.1.21) that catalyze the hydrolysis of cellobiose to glucose. The term “beta-glucosidase” is further defined herein as a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. Beta-glucosidase activity can be determined using any suitable method (See e.g., J. Basic Microbiol., 42: 55-66 [2002]). One unit of beta-glucosidase activity is defined as 1.0 pmole of p-nitrophenol produced per minute at 40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20.

As used herein, the term “glycoside hydrolase 61” and “GH61” refers to a category of cellulases that enhance cellulose hydrolysis when used in conjunction with one or more additional cellulases. The GH61 family of cellulases is described, for example, in the Carbohydrate Active Enzymes (CAZY) database (See e.g., Harris et al., Biochem., 49(15):3305-16 [2010]).

A “hemicellulase” as used herein, refers to a polypeptide that can catalyze hydrolysis of hemicellulose into small polysaccharides such as oligosaccharides, or monomeric saccharides. Hemicellulloses include xylan, glucuonoxylan, arabinoxylan, glucomannan and xyloglucan. Hemicellulases include, for example, the following: endoxylanases, beta-xylosidases, alpha-L-arabinofuranosidases, alpha-D-glucuronidases, feruloyl esterases, coumaroyl esterases, alpha-galactosidases, beta-galactosidases, beta-mannanases, and beta-mannosidases.

As used herein, the terms “xylan degrading activity” and “xylanolytic activity” are defined herein as a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl esterases) (See e.g., Biely and Puchard, J. Sci. Food Agr. 86:1636-1647 [2006]; Spanikova and Biely, FEBS Lett., 580:4597-4601 [2006]; and Herrmann et al., Biochem. J., 321:375-381 [1997]).

Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans. A common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan (See e.g., Bailey et al., J. Biotechnol., 23:257-270 [1992]). In some embodiments, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 mL reactions, 5 mg/mL substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay (See e.g., Lever, Anal. Biochem., 47:273-279 [1972]).

As used herein the term “xylanase activity” refers to a 1,4-beta-D-xylan-xylohydrolase activity (E.C. 3.2.1.8) that catalyzes the endo-hydrolysis of 1,4-beta-D-xylosidic linkages in xylans. In some embodiments, xylanase activity is determined using birchwood xylan as substrate. One unit of xylanase activity is defined as 1.0 μmole of reducing sugar (measured in glucose equivalents; See e.g., Lever, Anal. Biochem., 47:273-279 [1972]) produced per minute during the initial period of hydrolysis at 50° C., pH 5 from 2 g of birchwood xylan per liter as substrate in 50 mM sodium acetate containing 0.01% TWEEN® 20.

As used herein, the term “beta-xylosidase activity” refers to a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1→4)-xylooligosaccharides, to remove successive D-xylose residues from the non-reducing termini. In some embodiments of the present invention, one unit of beta-xylosidase activity is defined as 1.0 μmole of p-nitrophenol produced per minute at 40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20.



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Application #
US 20120270278 A1
Publish Date
10/25/2012
Document #
13539799
File Date
07/02/2012
USPTO Class
435100
Other USPTO Classes
435 72, 435155, 435160, 435165
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Chemistry: Molecular Biology And Microbiology   Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition   Preparing Compound Containing Saccharide Radical   Disaccharide