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Polypeptides of alicyclibacillus

A Bacillus, alicyclic acid technology, applied in the direction of peptides, depsipeptides, bacteria, etc.

Inactive Publication Date: 2013-12-04
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] A disadvantage of genome sequencing is that the vast majority of sequences obtained encode non-secreted proteins

Method used

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  • Polypeptides of alicyclibacillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0389] Example 1: Identification of functional polypeptides secreted by Alicyclobacillus DSM 15716

[0390] Genomic library construction

[0391] Chromosomal DNA of Alicyclobacillus DSM 15716 was prepared by using standard molecular biology techniques (Ausuble et al., 1995 "Current protocols in molecular biology", published by John Wiley and Sons). The prepared DNA was partially digested with Sau3A and separated on an agarose gel. Elute, pellet and resuspend the 3 to 8 kilobase fragments in an appropriate buffer.

[0392] By using Stratagene ZAP Express TM Predigested Vector Kit and StratageneZAP Express TM Predigested Gigapack Cloning kit (Bam HI predigested) (Stratagene Inc., USA) Genomic libraries were prepared according to the manufacturer's instructions / recommendations. The generated λZAP contained 38000 pfu, 10000 of which were selected for mass digestion. The resulting 70000 E. coli colonies were pooled and plasmids were prepared using the Qiagen Spin Mini prep k...

Embodiment 2

[0412] Example 2: Determination of function by homology

[0413] The functions of the polypeptides of SEQ ID NO: 26 to SEQ ID NO: 50 are annotated by comparison with gene or polypeptide sequences of known function. Polypeptides of the invention are compared to a list of most related sequences from public and internal contig databases. The BLASTX 2.Oa19MP-WashU [July 14, 1998] program was then used to compare the contigs (from which SEQ ID NO: 26 to SEQ ID NO: 50 were derived) to sequences available from standard public DNA and protein sequence databases. Careful analysis of sequence alignments of SEQ ID NO: 26 to SEQ ID NO: 40 with their most related functionally known sequences from other databases made it possible to predict the function of these polypeptides on the basis of the degree of amino acid identity. Even when the total amino acid identity is 40% (usually difficult to make a good prediction), we can predict SEQ ID NO: 26 to SEQ ID NO: 40 functions. When the amino...

Embodiment 3

[0414] Embodiment 3: Preparation of the polypeptide of SEQ ID NO: 26 to SEQ ID NO: 50

[0415] In order to prepare the polypeptide of SEQ ID NO: 26 to SEQ ID NO: 50, by combining the DNA encoding the open reading frame with a suitable host strain (such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis or Bacillus clausii or lipid Bacillus cycloacidum derivatives) were fused to express the genes encoding these polypeptides contained in SEQ ID NO: 1 to SEQ ID NO: 25. Promoters can be inducible or constitutive. Any signal sequence of SEQ ID NO: 26 to SEQ ID NO: 50 can be exchanged for an appropriate signal peptide from another bacterium. Expression constructs can be part of a plasmid or linear DNA. It can be integrated into the chromosome of the host strain by recombination or it can be present in the host cell as a plasmid. Transformed cells carrying the gene of interest are then cultured in the desired volume in an appropriate medium. If an inducible promoter i...

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Abstract

Isolated mature functional polypeptides are disclosed that are at least 90% identical to and perform the same function as the corresponding secreted polypeptide obtainable from the bacterium Alicyclobacillus deposited under accession number DSM 15716.

Description

technical field [0001] The present invention relates to functional and effective polypeptides encoded by polynucleotides contained in the genome of the bacterium Alicyclobacillus deposited under the accession number DSM 15716. The present invention also relates to polynucleotides encoding these polypeptides or promoting their expression, constructs of these polynucleotides and methods for preparing polypeptides. The present invention also relates to compositions comprising the polypeptide and polynucleotide and uses of the polypeptide. The present invention also relates to Alicyclobacillus bacteria deposited under the accession number DSM 15716. Background technique [0002] Some enzymes derived from Alicyclobacillus are known, for example Matzke et al.; Genecloning, nucleotide sequence and biochemical properties of a cytoplasmiccyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius ATCC 2700; reclassification of a group of enzymes, submitted (MAR-1999 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N9/42C12N9/52C12N9/24C12N9/90C12N15/53C12N15/55C12N15/61C07K14/32C12N9/16C12N1/21
Inventor R·惠尔廷S·F·拉森P·R·奥斯特伽德
Owner NOVOZYMES AS
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