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Polypeptides of Alicyclobacillus sp.

a technology of alicyclobacillus and polypeptides, which is applied in the field of functional polypeptides, can solve the problems of not being able to identify functional enzymes, limited to the availability of enzyme assays, and large majority of sequences obtained encode non-secreted proteins

Inactive Publication Date: 2005-07-07
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] The present inventors have found a strain of Alicyclobacillus namely Alicyclobacillus sp. DSM 15716 which grows at low pH (approx 4-5) and at high temperature (50-60° C.). This strain is interesting...

Problems solved by technology

This approach is limited to the availability of enzyme assays and does not allow the identification of functional enzymes or polypeptides for which the activity is still unknown.
However, only a few percent of a microorganisms' genome encodes secreted proteins.
One disadvantage of genome sequencing is that the vast majority of the obtained sequences encode non secreted proteins.

Method used

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  • Polypeptides of Alicyclobacillus sp.
  • Polypeptides of Alicyclobacillus sp.
  • Polypeptides of Alicyclobacillus sp.

Examples

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example 1

Identifying Functional Polypeptides Secreted by Alicyclobacillus sp. DSM 15716

Genomic Library Construction

[0252] Chromosomal DNA from Alicyclobacillus sp. DSM 15716 was prepared by using standard molecular biology techniques (Ausuble et al. 1995 “Current protocols in molecular biology” Publ: John Wiley and sons). The prepared DNA was partially cleaved with Sau3A and separated on an agarose gel. Fragments of 3 to 8 kilobases were eluted and precipitated and resuspended in a suitable buffer.

[0253] A genomic library was made by using the Stratagene ZAP Express™ predigested Vector kit and Stratagene ZAP Express™ predigested Gigapack® cloning kit (Bam HI predigested) (Stratagene Inc., USA) following the instructions / recommendations from the vendor. The resulting lambdaZAP library comprised 38000 pfu of which 10000 were collected for mass excision. The resulting 70000 E. coli colonies were pooled and plasmids were prepared by using the Qiagen Spin Mini prep kit (Qiagen, Germany). The ...

example 2

Determining Function by Homology

[0266] The function of the polypeptides SEQ ID NO: 26 to SEQ ID NO: 50 were annotated by sequences comparison with genes or polypeptides of known function. The polypeptides of the invention were compared to a list of closest related sequences from public and inhouse databases of contig's. The contigs, from which SEQ ID NO: 26 to SEQ ID NO: 50 were derived, were subsequently compared to sequences available in standard public DNA and protein sequences databases by using the program BLASTX 2.0a19 MP-WashU [14 Jul. 1998]. A careful analysis of sequence alignments of SEQ ID NO: 26 to SEQ ID NO: 40 to their closest related sequences with known function 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 overall amino acid identity was below 40%, which usually makes it difficult to make a good prediction, we were able to predict the function of SEQ ID NO: 26 to ...

example 3

Preparing Polypeptides of SEQ ID NO: 26 to SEQ ID NO: 50

[0267] To prepare the polypeptides of SEQ ID NO: 26 to SEQ ID NO: 50, the genes comprised in SEQ ID NO: 1 to SEQ ID NO: 25 encoding these polypeptides are expressed by fusing the DNA encoding the open reading frame to DNA a promoter, ribosome-binding site and terminator suitable for genes expression in an appropriate host strain, for example Escherichia coli, Bacillus subtilis, Bacillus licheniformis or Bacillus clausii or a derivative of Alicyclobacillus sp. The promoter can either be an inducible promotor or a constitutive promoter. Any signal sequences of SEQ ID NO: 26 to SEQ ID NO: 50 can be exchanged with a suitable signal peptide of another bacterium. The expression construct can either be part of a plasmid or of a linear DNA. It can be integrated into the chromosome of the host strain by recombination or it can be present in the host cell on a plasmid. Then the transformed cells carrying the gene of interest are grown i...

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Abstract

Isolated polypeptides are disclosed selected from the group consisting of: (a) polypeptides comprising an amino acid sequence which has at least 90% identity with a sequence of a mature polypeptide comprised in the group of SEQ ID NO: 26 to SEQ ID NO:50; (b) polypeptides which are encoded a nucleotide sequence which hybridize under high stringency conditions with a polynucleotide probe selected from the group consisting of (i) the complementary strand to a nucleotide sequence selected from the group of regions of SEQ ID NO: 1 to SEQ ID NO: 25 encoding a mature polypeptide. (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from the group of regions of SEQ ID NO: 1 to SEQ ID NO: 25 encoding a mature polypeptide wherein the polypeptides have a function of the corresponding mature polypeptides comprised in SEQ ID NO:26 to SEQ ID NO:50

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims, under 35 U.S.C. 119, priority or the benefit of Danish application Nos. PA 2004 00010, filed Jan. 6, 2004, and PA 2004 00165, filed Feb. 4, 2004, filed, the contents of which are fully incorporated herein by reference. FIELD OF THE INVENTION [0002] The present invention relates to functional polypeptides encoded by polynucleotides comprised in the genome of Alicyclobacillus sp. deposited under deposit accession number DSM 15716. The invention relates further to the polynucleotides and constructs of such polynucleotides encoding such polypeptides or facilitating their expression as well as to method for preparing the polypeptide. Still further the invention relates to compositions comprising the polypeptide and to uses of the polypeptide. BACKGROUND OF THE INVENTION [0003] Some enzymes from the genus of Alicyclobacillus species are known such as described in Matzke et al.; Gene cloning, nucleotide sequence and bi...

Claims

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Application Information

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IPC IPC(8): A23L35/00C07K14/32C12N9/02C12N9/24C12N9/42C12N9/52C12N9/90
CPCC07K14/195C07K14/32C12N9/0004C12N9/52C12N9/90C12R1/01C12Y301/01058C12N9/2402C12N9/2437C12N9/244C12Y305/02006C12N9/18C12N9/86C12Y302/01096A61P35/00C12N1/205C12R2001/01
Inventor WILTING, REINHARDLASSEN, SORENOSTERGAARD, PETER
Owner NOVOZYMES AS
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