Midwest Research Institute

Alliance for Sustainable Energy, LLC

William S Adney

Michael E Himmel

Stephen Robert Decker

Todd B Vinzant

Shi-You Ding

John O Baker

Yat-Chen Chou

Gregg Tyler Beckham

Eric E Jarvis

Melvin P Tucker, Iii

Jeffrey G Linger

Arjun Singh

Steven R Thomas

Qi Xu

Kara Podkaminer

Larry Taylor, Ii

Rafael A Nieves

Karel Grohmann

Mark R Nimlos

Eric P Knoshaug

Shih-You Ding

Suzanne Lantz McCarter

Suzanne Mc Carter

Raphael Nieves

Michael F Crowley

Tina Jeoh

Nucleic acid sequences encoding chimeric polypeptides that exhibit enhanced cellulase activities are disclosed herein. These nucleic acids may be expressed in hosts such as fungi, which in turn may be cultured to produce chimeric polypeptides. Also disclosed are chimeric polypeptides and their use in the degradation of cellulosic materials.

Enhanced processive cellulases

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

Cellobiohydrolase I enzymes

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes fromdemonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase fromand 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-nativehost. In a specific example, the specific performance of the formulation using purified recombinant Cel7A fromexpressed inis increased by more than 200% when compared to a formulation using purified Cel7A from

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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, and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized fromand, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

Thermal tolerant avicelase from Acidothermus cellulolyticus

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A) enzymes fromdemonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase fromand 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-nativehost. In a specific example, the specific performance of the formulation using purified recombinant Cel7A fromexpressed inis increased by more than 200% when compared to a formulation using purified Cel7A from

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. 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.

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.

Cellobiohydrolase I gene and improved variants

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. 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.

Thermal tolerant mannanase 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 fromThe invention further provides recombinant forms of the identified GuxA. Methods of making and using GuxA polypeptides, including fusions, variants, and derivatives, are also disclosed.

A process for production of an alkaline tolerant dextranase enzyme comprises culturing a dextran-producing microorganism 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.

Alkaline tolerant dextranase from streptomyces anulatus

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.

Method for increasing thermostability in cellulase ennzymes

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.

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Golden, CO, United States of America

William S Adney