Beta-glucosidase from neurospora crassa

A technology of glucosidase and hemicellulase, applied in the direction of glycosylase, enzyme, hydrolase, etc., can solve the problems of toxicity and low efficiency of crystalline cellulose

Inactive Publication Date: 2015-07-29
DANISCO US INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

β-glucosidase plays an important role in the release of glucose from cellooligosaccharides such as cellobiose, which is toxic to microorganisms such as yeast used to ferment sugar to ethanol; and cellobiose also inhibits endoglucose Activity of carbohydrases and cellobiohydrolases such that they are ineffective in further hydrolyzing crystalline cellulose

Method used

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  • Beta-glucosidase from neurospora crassa
  • Beta-glucosidase from neurospora crassa
  • Beta-glucosidase from neurospora crassa

Examples

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example

[0224] The following examples are provided to demonstrate and illustrate certain preferred embodiments and aspects of the present disclosure and should not be construed as limiting.

example 1

[0226] 1-A. Cloning and Expression of Gene Expression of Nc3A and Benchmark Trichoderma reesei Bgl1

[0227] 1-A-a. Construction of Trichoderma reesei bgl1 expression vector

[0228] The N-terminal portion of the native T. reesei β-glucosidase gene bgl1 was codon optimized (DNA 2.0, Menlo Park, CA). This synthetic portion contained the first 447 bases of the enzyme coding region. This fragment was subsequently amplified by PCR using primers SK943 and SK941 (see below). Using primers SK940 and SK942 (see below), the remaining region of the native bgl1 gene was PCR amplified from a genomic DNA sample extracted from Trichoderma reesei strain RL-P37 (Sheir-Neiss, G et al., (1984) Appl . Microbiol. Biotechnol. 20:46-53 (Sheir-Neiss, G et al., 1984, Applied Microbiology and Biotechnology, Vol. 20, pp. 46-53)). These two PCR fragments of the bgl1 gene were fused together in a fusion PCR reaction using primers SK943 and SK942 as follows:

[0229] Forward primer SK943: (5'-CACC...

example 2

[0256] Example 2: Various Assays

[0257] 2-A. Protein concentration measurement by UPLC

[0258] Protein quantification was performed using an Agilent HPLC 1290 Infinity system and a Waters ACQUITY UPLC BEHC4 column (1.7 μm, 1×50 mm). Using a six minute program using an initial gradient from 5% to 33% acetonitrile (Sigma-Aldrich) in 0.5 minutes, followed by a gradient from 33% to 48% in 4.5 minutes, This was followed by a step gradient to a final 90% acetonitrile. A protein standard curve based on purified T. reesei Bgl1 was used to quantify the Nc3A polypeptide.

[0259] 2-B. Chloro-nitro-phenyl-glucoside (CNPG) hydrolysis assay

[0260] Two hundred (200) μL of 50 mM sodium acetate buffer, pH 5, was added to each well of the microtiter plate. Five (5) μL of enzyme diluted in 50 mM sodium acetate buffer (pH 5) was also added to each well. The plate was covered and allowed to equilibrate for 15 minutes at 37°C in an Eppendorf thermomixer. Twenty (20) μL of 2 mM 2-c...

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Abstract

The present compositions and methods relate to a beta-glucosidase from Neurospora crassa, polynucleotides encoding the beta-glucosidase, and methods of make and / or use thereof. Formulations containing the beta-glucosidase are suitable for use in hydrolyzing lignocellulosic biomass substrates.

Description

[0001] priority [0002] This application claims priority to US Provisional Application Serial No. 61 / 720,745, filed October 31, 2012, which is hereby incorporated by reference in its entirety. technical field [0003] The compositions and methods of the present invention relate to beta-glucosidase polypeptides obtainable from Neurospora crassa, polynucleotides encoding beta-glucosidase polypeptides, and methods of making and using the same. Formulations and compositions comprising β-glucosidase polypeptides can be used to degrade or hydrolyze lignocellulosic biomass. Background technique [0004] Cellulose and hemicellulose are the most abundant plant materials produced through photosynthesis. They can be degraded and used as energy sources by a variety of microorganisms (eg, bacteria, yeast, and fungi) that produce extracellular enzymes capable of hydrolyzing polymeric substrates into monomeric sugars (Aro et al., (2001) J. Biol .Chem., 276:24309-24314 (Aro et al., 2001...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42
CPCC12N9/2445C12Y302/01021C12P19/02C12P19/14
Inventor B·S·鲍尔M·K·福达拉
Owner DANISCO US INC
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