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Publication Number
US-10774318-B2
Publication Date
2020-09-15
Expiration Date
Abstract
An object is to identify endoglucanase and β-glucosidase genes by isolating genomic DNA containing cellulase genes, which are classified into endoglucanases or β-glucosidases, from Acremonium cellulolyticus, and sequencing the nucleotide sequences thereof. The inventors intensively compared the amino acid sequences of known endoglucanases and β-glucosidases with each other to find conserved region of amino acid sequences in Acremonium cellulolyticus, and various primers were designed based on the information. PCR was carried out using the various primers thus designed and genomic DNA or cDNA as a template. As a result, gene fragments of endoglucanases and β-glucosidases were obtained. Primers were designed based on the gene fragments, and PCR was carried out to amplify nine genes of endoglucanases and β-glucosidases. The nucleotide sequences thereof were sequenced, and the present invention was completed.
Core Innovation
The invention provides identification and cloning of multiple endoglucanase genes (ACC3, ACC5-ACC10) and β-glucosidase genes (BGLC, BGLD) from Acremonium cellulolyticus. The genes are identified using conserved-region sequence comparisons, primer design, PCR including inverse PCR, and sequencing. Recovered gene fragments are used to deduce ORF-based amino acid sequences and corresponding DNA sequences.
The document reports ORF-based amino acid sequence deduction for the endoglucanase and β-glucosidase gene sequences, with reference to SEQ ID NOs and predicted intron positions and signal peptide predictions. It further discloses that corresponding DNA sequences are used to form expression vectors and to introduce the DNA into transformed hosts or host cells, including plasmids corresponding to ACC3, ACC5-ACC10, BGLC, and BGLD.
The disclosure centers on recombinant production of cellulase components, including endoglucanase and β-glucosidase, and on using such components in cellulase preparations to improve cellulose saccharification. The document also asserts industrial uses connected to biomass saccharification and downstream applications including paper pulp water freeness, animal feed digestibility, and deinking waste paper.
Claims Coverage
The partial content provides two independent claims, centered on an endoglucanase protein meeting a defined sequence identity and substitution-modification requirement, and a cellulase preparation containing that protein or a protein comprising amino acids 1-376 of SEQ ID NO: 8 together with an added preservative. The inventive features are grounded in sequence identity to SEQ ID NO: 8 (amino acids 1-376), a substitution modification requirement, and the inclusion of an effective amount of an added preservative.
Endoglucanase protein with high identity to SEQ ID NO: 8 and substitution modification
A protein having endoglucanase activity, having at least 98% sequence identity to amino acids 1-376 of SEQ ID NO: 8, and containing at least one substitution modification relative to amino acids 1-376 of SEQ ID NO: 8.
Cellulase preparation with defined endoglucanase component and added preservative
A cellulase preparation comprising a protein having endoglucanase activity with at least 98% sequence identity to amino acids 1-376 of SEQ ID NO: 8 and containing at least one substitution modification relative to amino acids 1-376 of SEQ ID NO: 8, or a protein comprising amino acids 1-376 of SEQ ID NO: 8, wherein said cellulase preparation comprises an effective amount of an added preservative.
Across the provided independent claims, the core sequence-based invention is an endoglucanase protein defined by at least 98% sequence identity to amino acids 1-376 of SEQ ID NO: 8 and at least one substitution modification, optionally instantiated as the amino-acid 1-376 sequence itself in a cellulase preparation. The preparation claim additionally requires an effective amount of an added preservative.
Stated Advantages
Improving cellulose saccharification.
Documented Applications
Biomass saccharification.
Paper pulp water freeness.
Animal feed digestibility.
Deinking waste paper.
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