Production of proteinase from gene recombinant pichia stipitis

A technology of Pichia pastoris and gene recombination, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, genetic engineering, etc., and can solve the problem that the expression product has no biological activity, etc.

Inactive Publication Date: 2009-06-10
YUNNAN NORMAL UNIV
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Problems solved by technology

However, the expressed protein failed to be secreted outside the cell, but accumulated in the cell in the form of protease precursor. When the signal peptide sequence of yeast invertase was integrated into the upstream of mature nprE, neutral protease was secreted outside the cell, however , the expression product has no biological activity due to high glycosylation

Method used

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  • Production of proteinase from gene recombinant pichia stipitis
  • Production of proteinase from gene recombinant pichia stipitis
  • Production of proteinase from gene recombinant pichia stipitis

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Experimental program
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Embodiment

[0051] Prepare neutral protease as follows:

[0052] 1. Construction of engineering bacteria

[0053] Using the method of polymerase chain reaction (PCR), the complete sequence of the gene encoding neutral protease was cloned from Bacillus subtilus 168, and then cloned into the Pichia pastoris expression vector PGAP to obtain recombinants, which were respectively transformed into Pichia pastoris expression hosts GS115, SMD1168, and then screen out constitutive high-expression transformants (GSp5 and SMDp18) respectively (the neutral protease can be produced in ordinary glucose medium). 从Bacillus subtilus 168中所克隆出的基因片断具备编码下列氨基酸序列:MGLGKKLSVRVAASFMSLSISLPGVQAAEGHQLKENQTNFLSKKPIAQSELSAPNDKAVKQFLKKNSNIFKGDPSKSVKLVESTTDALGYKHFRYAPVVNGVPIKDSQVIVHVDKSDNVYAVNGELHNQSAAKTDNSQKVSSEKALALAFKAIGKSPDAVSNGAAKNSNKAELKAIETKDGSYRLAYDVTIRYVEPEPANWEVLVDAETGSILKQQNKVEHAAATGSGTTLKGATVPLNISYEGGKYVLRDLSKPTGTQIITYDLQNRQSRLPGTLVSSTTKTFTSSSQRAAVDAHYNLGKVYDYFYSNFKRNSYDNKGSKIVSSVHYGTQYNNAAWTGDQMIYGDGDGSFFSPL...

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Abstract

The invention is a method that producing neutral proteinase by a gene recombination Pichia.The character is producing neutral proteinase by Pichia pastoris. Using Pichia pastoris to express extraneous source protein has many advantages such as better expression, better stability, and stronger secretion. As Pichia pastoris can not synthesize Bacillus subtilis proteinase itself,this invention carried Bacillus subtilis proteinase gene into Pichia pastoris to help producing much more Bacillus subtilis proteinase.Because Pichia pastoris is easy to ferment in high density and it has highly secrection, it is easy to industrial produce cheap Bacillus subtilis proteinase.

Description

technical field [0001] The invention relates to a method for producing neutral protease by genetically recombined Pichia pastoris. Background technique [0002] Protease is a type of enzyme that catalyzes the hydrolysis of peptide bonds. Neutral protease refers to a type of protease whose optimum pH is 6.0-7.5. For food industry etc. [0003] The research and development of neutral protease has been started since the 1970s, and industrialization was realized at home and abroad in the early 1980s. Most of the produced strains use Aspergillus for solid fermentation, mainly Aspergillus and rice Aspergillus. Bacteria are mostly used in liquid fermentation, among which Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus have higher yields. Liquid fermentation is also produced using actinomycetes. At present, the production of neutral protease in my country mainly uses Aspergillus aeruginosa and Bacillus subtilis, but the fermentation unit of neutral protease has...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50C12N9/60C12N1/19C12N15/57C12N15/81C12P21/02C12R1/84
Inventor 黄遵锡许波唐湘华
Owner YUNNAN NORMAL UNIV
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