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Thermostable C. BESCII enzymes

A kind of furanase, xylosidase technology, applied in the direction of enzyme, hydrolase, glycosylase, etc., can solve problems such as unavailability

Active Publication Date: 2014-08-27
THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Currently, the microorganisms used to ferment sugars into biofuels such as ethanol cannot utilize complex polysaccharides such as cellulose and hemicellulose
As a result, there is a major bottleneck in the conversion of lignocellulosic materials into biofuels

Method used

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  • Thermostable C. BESCII enzymes
  • Thermostable C. BESCII enzymes
  • Thermostable C. BESCII enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0551] Example 1: Endoxylanase Cb193 (SEQ ID NOs: 3 and 4)

[0552]The endoxylanase Cb193 was identified in Caldicellulosiruptor bescii. This enzyme is the gene product of Cb193, where Cb stands for C. bescii. This endoxylanase randomly cleaves the xylose backbone of hemicellulose to generate short linkages of xylose on the β-1,4-linkages. These xylooligosaccharides may comprise two or more sugar subunits. The Cb193 protein is 671 amino acids long with a molecular weight of 77.7 kDa (His-tag + truncated Cb193 protein). The protein has two putative carbohydrate-binding modules (CBMs) inserted within the glycoside hydrolase (GH) family 10 catalytic domain (see Figure 2A ).

[0553] Cloning of Cb193

[0554] The gene for Cb193 was PCR amplified from Caldicellulosiruptor bescii genomic DNA using iProof HF DNA polymerase (BIO-RAD). Cb193 was amplified using the following primer pair:

[0555] Cb193 positive

[0556] 5'-GACGACGACAAGATGAACTTTGAAGGAAGAGAC-3' (SEQ ID NO: 134...

Embodiment 2

[0589] Example 2: Endoxylanase Cb195 (SEQ ID NOs: 7 and 8)

[0590] An endoxylanase Cb195 was identified in Caldicellulosiruptor bescii. This enzyme is the gene product of Cb195, where Cb stands for C. bescii. This endoxylanase randomly cleaves the xylose backbone of hemicellulose to generate shorter chains at the β-1,4-linkages. These xylo-oligosaccharides can range from containing two or more saccharide units. The Cbl95 protein is 351 amino acids long and has a molecular weight of 41.9 kDa (histidine tag + Cbl95 protein) (Figure 2).

[0591] Cloning of Cb195

[0592] The Cb195 gene (BIO-RAD) was amplified from Caldicellulosiruptor bescii genomic DNA by PCR using iProof high-frequency DNA polymerase.

[0593] The PCR mix contains the following:

[0594] PCR reaction

[0595]

[0596] To amplify genes from genomic DNA, the following PCR cycles were used.

[0597] PCR protocol

[0598]

[0599] After the PCR amplification described above, the amplification of the ...

Embodiment 3

[0619] An α-L-arabinofuranosidase was identified in Caldicellulosiruptor bescii. This enzyme is the gene product of Cb1172. α-L-arabinofuranosidase cleaves arabinose groups from the xylose backbone or from branched or debranched chains. The Cb1172 protein is 505 amino acids in length and has a molecular weight of 59.6 kDa (His tag + Cb1172 protein). The protein has a glycoside hydrolase (GH) family 51 catalytic domain ( Figure 6D ).

[0620] Clone Cb1172

[0621] Use iProof TM High-Fidelity DNA polymerase (BIO-RAD) amplifies the Cb1172 gene from Caldicellulosiruptor bescii DSM6725T genomic DNA by PCR. The Cb1172 gene was amplified with the following primer set.

[0622] Cb1172 forward primer

[0623] 5'-GAC GAC GAC AAG ATG AAA AAA GCA AAA GTC ATC TAC-3' (SEQ ID NO: 136)

[0624] Cb1172 reverse primer

[0625] 5'-GAG GAG AAG CCC GGT TAA TTT TCT TTC TTC TTT AAC CTG-3' (SEQ ID NO: 137)

[0626] The PCR mix contains the following:

[0627] PCR reaction

[0628]

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Abstract

The disclosure provides thermostable enzymes isolated from Caldicellulosiruptor bescii and fragments thereof useful for the degradation of cellulose and / or hemicellulose, including thermostable cellulases and hemicellulases. The disclosure further provides nucleic acids encoding the thermostable enzymes of the disclosure. The disclosure also provides methods for the conversion of cellulose and hemicellulose into fermentable sugars using thermostable enzymes of the disclosure. The disclosure also provides enzyme cocktails containing multiple enzymes disclosed herein. The enzymes can be used to release sugars present in cellulose or hemicellulose for subsequent fermentation to produce value-added products.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application 61 / 425,623, filed December 21, 2010, and US Provisional Application 61 / 532,060, filed September 7, 2011, the contents of which are hereby incorporated by reference in their entirety. technical field [0003] The present invention relates to compositions and methods for degrading cellulose, hemicellulose, and materials containing cellulose and / or hemicellulose. In particular, the disclosure provides thermostable enzymes for degrading cellulose, nucleic acids encoding the enzymes, and methods of use thereof. The disclosure also provides thermostable enzymes for degrading hemicellulose, nucleic acids encoding the enzymes, and methods of use thereof. The present invention further provides a thermostable enzyme that increases the activity of thermostable cellulase and / or hemicellulase, a nucleic acid encoding the enzyme, and a method of use thereof. [0004] Seq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P1/04C07K14/195
CPCC12N9/2402C12P7/14C12P19/14C12P7/16C12N9/248C12N9/2482C12P7/04C12Y302/01055C12N9/2434C12N9/18C12Y302/01072C12N9/2405C12Y302/01008C12Y302/01139C12Y301/01072C12P19/02Y02E50/10
Inventor 韩业君文永焕苏小运吉田章介宫城笃志迪伦·多德罗德里克·I.·麦克凯艾萨克·K.O.·坎恩
Owner THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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