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Microbial swollenin protein, DNA sequences encoding such swollenins and method of producing such swoolenins

A technology of expansin and protein, applied in the field of Trichoderma species, can solve the problems that the effectiveness and operability of apple penicillin are in their infancy, and the large-scale production of expression system is not ideal.

Inactive Publication Date: 2000-09-27
GENENCOR INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Despite previous pioneering work on cell wall extension and its causes, work on the effectiveness and operability of apple penicillins is still in its infancy
In addition, the source of apple penicillin so far is only from plants, and its expression system may not be ideal for large-scale production

Method used

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  • Microbial swollenin protein, DNA sequences encoding such swollenins and method of producing such swoolenins
  • Microbial swollenin protein, DNA sequences encoding such swollenins and method of producing such swoolenins
  • Microbial swollenin protein, DNA sequences encoding such swollenins and method of producing such swoolenins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Encoding the new swellin trichoderma (longibrachiatum) cDNA clone

[0065] Figure 1 shows the nucleotide (SEQ ID NO: 1) and deduced corresponding amino acid sequence (SEQ ID NO: 2), as described by Saloheimo et al., 1994, Molec. Microbiol., 13:219-228. The cDNA exhibits the following properties that help describe the gene:

[0066] An open reading frame of 1482 nt was identified and the encoded amino acids were deduced.

[0067] The first 18 amino acids of the putative protein have the following expected properties of the secretion signal sequence and signal cleavage site. A positively charged amino acid (lysine) is adjacent to the amino-terminal methionine followed by a hydrophobic amino acid sequence and an apparent signal peptidase cleavage site after amino acid Ile18. The putative N-terminus of mature swollenin is Gln-Gln. Similarly, many mature cellulases from Trichoderma have glutamine residues at their N-terminus (e.g., CBHI, CBHII, EGI, EGII, and E...

Embodiment 2

[0077] In order to investigate the regulation of the expression of the expansin gene in Trichoderma, the following experiments were carried out. Longlbrachiatum strain QM9414 was grown for three days in minimal medium (Penttila et al., 1987, Gene, 61: 155-164) in shaker flasks (28°C, 200 rpm). The medium contained 5% glucose or 2% cellulose. To determine sophorose induction, the strains were grown for three days in minimal medium containing 2% sorbitol, after which sophorose was added until a final concentration of 1 mM was reached. The cultivation was carried out for another 10 hours and the same amount of sophorose was added. The incubation was terminated 5 hours after the second addition. Cultured in 2% sorbitol without adding sophorose for 87 hours was used as a control. After cultivation, the mycelium was harvested by filtration through a glass fiber filter, washed with 0.9% NaCl and frozen. Total RNA was isolated from mycelial samples according to the method of Chirg...

Embodiment 3

[0087] Given the exemplary swellin gene provided above, it is routine for one of ordinary skill in the art to clone the T. miguro swellin gene from genomic DNA or cDNA libraries by colony hybridization using the PCR fragment inserted into pSLexpPCR as a probe . Example 3 Cloning of Trichoderma mihei Expansin Genome Copy and Its Expression in A.niger var.awamori

[0088] Cloning of a genomic copy of T. miehiro expansin by PCR. The template DNA was obtained from Trichoderma miera RutC-30 (ATCC 56765), and the primers corresponding to the 5' and 3' ends of the swollenin coding region were named GCI-PVS-055 (gcg cag atc tca gca atg gct ggt aag ctt atc ctc g) and GCI-PVS-056 (gcg ctc tag atc aat tct ggc taa act gca cac c).

[0089] The PCR amplified fragment was digested with BglII and XbaI and cloned into BglII-XbaI cut pGAPT-PT to form pGAPT-expC. Sequencing of the insert revealed that the chromosomal copy of the expansin gene has five introns.

[0090] A chromosomal copy of ...

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Abstract

A novel microbial protein is described which appears to have significant homology to plant expansin proteins and has the ability to weaken filter paper and swell cellulose. A DNA is described which encodes the novel protein.

Description

Background of the invention [0001] Osmotic uptake of water is the driving force for plant cell expansion. When water enters the cell, the protoplast expands, but is confined by the cell wall. In addition, rigid complexes of cellulose microfibrillar polymers embedded in a gelatinous matrix of pectin, hemicellulose, and proteins form part of the cell wall in mature cells. It has long been thought that there must be some "wall relaxing" factor that alters the mechanical properties of the immature cell wall to enable the elongation process. McQueen-Mason et al., Plant cell, Vol. 4, pp. 1425-1433 (1992) studied the regulation of plant cell enlargement by using a reconstitution approach. These authors found that crude protein extracts from the cell walls of growing cucumber seedlings had the ability to induce ex vivo cell wall expansion. Sequential HPLC fractionation of active wall extracts revealed two proteins with molecular weights of 29 and 30 kD associated with activity. Ea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A23K1/00A23K1/16A23K1/165C07K14/37C11D3/386C12N1/15C12N1/21C12N9/42C12N15/31C12P19/14C12P21/02C12R1/645D06M16/00D21C5/00D21H11/20D21H17/00D21H21/14
CPCD06M16/003C11D3/386D21H21/14C07K14/37A23K1/007D21H17/005D06M2101/06A23K1/1631A23K10/12A23K20/147
Inventor B·斯万森M·沃尔德M·潘迪拉J·彼尔M·萨罗海姆
Owner GENENCOR INT INC
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