Methods for improving by-products from fermentation processes using xylanase
A technology for xylanase and xylanase activity, which is applied in the field of improving by-products in the fermentation process and can solve problems such as differences in xylanase functions
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[0669] Nucleotide sequence preparation
[0670] Nucleotide sequences encoding proteins having specific properties as defined herein or proteins suitable for modification may be identified and / or isolated and / or purified from any cell or organism producing said protein. Various methods are well known in the art of nucleotide sequence identification and / or isolation and / or purification. By way of example, once suitable sequences have been identified and / or isolated and / or purified, PCR amplification techniques can be used to prepare further sequences.
[0671] By way of another example, genomic DNA and / or cDNA libraries can be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme is known, labeled oligonucleotide probes can be synthesized and used to identify enzyme-encoding clones from genomic libraries prepared from the organism. Alternatively, a labeled oligonucleotide probe containing a sequenc...
Embodiment 1
[0845] Materials and methods
[0846] Plasmid and library construction
[0847] A DNA sequence comprising the coding region of xylanase 4 (family GH10) FveXyn4 from the filamentous fungus Fusarium verticilloides was amplified from genomic DNA with gene-specific primers and extended with attB1 and attB2 sites, thereby Make BP was recombinantly cloned into pDonor221 vector (Invitrogen, USA). Suppliers BaseClear (Netherlands) and Geneart GmbH (Germany) will as Figure 20 The indicated pEntry-FveXyn4 plasmid was used as a template for the construction of combinatorial libraries.
[0848] Variants of FveXyn4 were generated as combinatorial libraries or by introducing specific mutations and were designed to include different numbers and combinations of the mutations shown in Table 1. Included among these variants are variants A, B, C, D and E.
[0849] Using the destination vector pTTTpyr2 ( Figure 21 )via Combinatorial variants were generated by recombinant technology...
Embodiment 2
[0902] Cloning of main chain (parent) xylanase (FveXyn4) from Fusarium moniliforme
[0903]Genomic DNA isolated from the F. moniliforme strain was used to amplify the xylanase gene. The sequence of the cloned gene (referred to as FveXyn4 gene) is shown in SEQ ID No.2. The mature protein encoded by the FveXyn4 gene is shown as SEQ ID No.1. Based on a PFAM search (http: / / pfam.sanger.ac.uk / ), the protein product of the gene FveXyn4 belongs to the glycosyl hydrolase family 10 (GH10).
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