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A fungal protease and use thereof

A serine protease, fungus technology, applied in the direction of hydrolase, biochemical treatment of enzymes/microorganisms, introduction of foreign genetic material using vectors, etc., can solve problems such as unresearched

Active Publication Date: 2015-04-01
AB ENZYMES GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Their use in other biotechnological processes remains unstudied

Method used

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  • A fungal protease and use thereof
  • A fungal protease and use thereof
  • A fungal protease and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0209] Example 1. Trichoderma reesei QM6a prb1 ( Tr prb1 ) And Fusarium graminearum ALKO1726 Fg prtS8A Cloning of protease gene

[0210] (A) Molecular biology methods for DNA isolation and use

[0211] Standard molecular biology methods are used for DNA isolation and enzymatic treatment (eg isolation of plasmid DNA, digestion of DNA to produce DNA fragments), transformation in E. coli, sequencing, etc. The basic method used is as described by the manufacturer of the enzyme, reagent or reagent expression kit, or as described in standard molecular biology manuals such as Sambrook and Russell (2001). The isolation of genomic DNA from Trichoderma reesei QM6a and Fusarium graminearum ALKO1726 was completed as described by Raeder and Broda (1985). A PhaseLock tube (Eppendorf, Germany) was used to perform the phenol extraction stage.

[0212] (B) Oligonucleotide primers for gene cloning

[0213] Using Trichoderma reesei QM6a and Fusarium graminearum ALKO1726 genomic DNA preparations as ...

Embodiment 2

[0223] Example 2. Production of Tr Prb1 and Fg_ALKO1726 recombinant proteases in Trichoderma reesei

[0224] (A) Preparation of expression cassette and its transformation into Trichoderma reesei

[0225] By separately amdS ( The acetamidase) marker gene was ligated into the plasmids pALK2650 and pALK2707 (Example 1c) to construct the expression plasmid pALK2701 (for the production of recombinant Tr Prb1 and Fg_ALKO1726 proteins in Trichoderma reesei). Tr prb1 ) And pALK2708 ( Fg prtS8A ). amdS Mark at cbh1 The terminator is then connected to the expression construct. Similar constructions have been described in, for example, Paloheimo et al. (2003). In the expression cassettes pALK2701 and pALK2708 ( image 3 with 4 ), through PCR will have its own signal sequence Tr prb1 with Fg prtS8A Genes and Trichoderma reesei cbh1 (cel7A) The promoter is exactly fused. Use generated after the stop codon in PCR Bam HI site, connect the 3-' end of the gene with cbh1 Terminator fusion...

Embodiment 3

[0231] Example 3. Purification and characterization of recombinant Tr Prb1 and Fg_ALKO1726 protease

[0232] Cells and solids were removed from the depleted medium obtained from the fermentation (Example 2) by centrifugation at 4°C for 30 minutes, 50,000 g (Sorvall RC6 plus). 15 ml of supernatant was used for the purification of protease. All purification steps are performed in the cold room. After centrifugation, the sample was filtered through a 0.44 μm filter (MILLEX HV Millipore) before being applied to a HiPrep 26 / 10 Desalting column (from GE Healthcare) equilibrated in 20 mM Tris pH 8.8. The gel-filtered samples were applied to a 20 mL Q Sepharose FF column (from GE Healthcare) equilibrated in 20 mM Tris pH 8.8. An Amicon Ultra centrifugal filter unit 10000 MWCO (Millipore) was used to concentrate the effluent fraction with proteolytic activity. The concentrated sample was applied to a Superdex 75 10 / 300 GL column (GE Healthcare) and eluted with 20 mM Hepes, 150 mM NaCl ...

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Abstract

The present invention is related to a fungal serine protease enzyme useful in modification, degradation or removal of proteinaceous material, which enzyme comprises an amino acid sequence of the mature Tr Prb1 enzyme having an amino acid sequence of SEQ ID NO: 10 or a variant thereof having similar activity. The serine protease is obtainable from Trichoderma. Also disclosed are nucleic acid sequences encoding said protease, such as plasmid pALK2650 comprising the nucleotide sequence SEQ ID NO: 10 of the full length enzyme deposited in E. coli RF8052 under accession number DSM 22635. Said protease is useful as an enzyme preparation applicable in detergent compositions and for treating fibers, for treating wool, for treating hair, for treating leather, for treating food or feed, or for any applications involving modification, degradation or removal of proteinaceous material at low or moderate temperature ranges.

Description

Invention field [0001] The present invention relates to fungal serine proteases useful in various industrial applications, especially in laundry and dishwashing detergents, wherein the performance of the enzyme at low or medium temperature ranges is advantageous. The present invention relates to isolated nucleic acid molecules encoding the enzymes, recombinant vectors, host cells for producing the enzymes, enzyme compositions containing the enzymes, and methods for preparing such compositions. The present invention also relates to various uses of the enzyme or the composition containing the enzyme. Background technique [0002] Microbial extracellular proteases account for the majority, and more than one-third of the total sales of industrial enzymes worldwide (Cherry and Fidantsef, 2003). About 90% of commercial proteases are detergent enzymes (Gupta et al., 2002). Other applications include, for example, food, feed, leather, medicine, diagnostics, waste management, and silver...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/37C12N15/11C12P21/02C12R1/00C12R1/885A23L29/00
CPCC11D3/386C12N9/58C11D3/38681A23L29/06C12N15/52C12N15/80D06M16/003
Inventor L.瓦尔塔卡里K.容图南S.马基南P.奥贾帕洛M.帕罗海莫
Owner AB ENZYMES GMBH
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