Construction method of recombinant bacillus subtilis of high-yield pullulanase

A technology of Bacillus subtilis and pullulanase, which is applied in the field of construction of recombinant Bacillus subtilis and can solve problems such as no resistance

Pending Publication Date: 2017-08-18
BAIYIN SINO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In order to improve the genetic stability of production strains, exogenous expression is usually integrated into the genome of the host cell, and antibiotic genes are often used as selection markers, but in the food industry, recombinant strains carrying antibiotic genes will bring biosafety Therefore, it is necessary to remove the antibiotic gene through secondary recombination or specific recombination after the integration of the foreign gene to obtain a non-resistant production strain

Method used

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  • Construction method of recombinant bacillus subtilis of high-yield pullulanase
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  • Construction method of recombinant bacillus subtilis of high-yield pullulanase

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Embodiment 1

[0388] Embodiment 1: Construction of recombinant Bacillus subtilis CNP2

[0389] 1. Obtaining integrated DNA

[0390] There are two operons expressed by pullulanase, the first pulB1 Including artificial tandem promoter Pamy-cryIII, codon-optimized signal peptide sequence SPamyL secreted by amylase, and codon-optimized pullulanase coding gene pulB , pulB The sequence is shown in SEQ ID NO. 3, pulB1 The sequence is shown in SEQ ID NO. 10; the second pulB2 Including artificial tandem promoter PxylA-cryIII, codon-optimized chitinase secreted signal peptide sequence SPchit and codon-optimized pullulanase coding gene pulB , pulB2 See SEQ ID NO. 11 for the sequence.

[0391] for apr E Gene expression cassette of knockout recombination arm and erythromycin resistance fragment apr-erm-apr As shown in SEQ ID NO. 8, for amyE Gene expression cassette of knockout recombination arm and kanamycin resistance fragment amy-kan-amy As shown in SEQID NO. 9.

[0392] Bundle ...

Embodiment 2

[0417] Embodiment 2: the fermentative production enzyme of recombinant Bacillus subtilis CNP2

[0418]Use recombinant Bacillus subtilis CNP2 as the production strain for shake flask fermentation: pick up recombinant Bacillus subtilis CNP2 with an inoculation loop and inoculate it into a 200 mL baffle bottle containing 25 mL liquid medium, 37°C, 200 r / min Fermentation culture is carried out, wherein the main components of the medium are corn steep liquor, maltose syrup, glucose, yeast powder and inorganic salt ions. After 20 hours of fermentation, samples were taken to measure the enzyme activity. The method for the determination of pullulanase activity was as follows: take 1.0 mL of acetic acid-sodium acetate buffer solution with pH=4.5 to prepare enzyme samples or diluted samples (the control group was samples inactivated in a boiling water bath), Add it to 1.0 mL of 0.5% pullulan solution, bathe in a water bath at 60°C for 30 min, then add 3.0 mL of 3,5-dinitrosalicylic acid...

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Abstract

The invention discloses a construction method of recombinant bacillus subtilis of high-yield pullulanase. The method comprises the steps that acid and heat resistance pullulanase gene is optimized by a codon, then is spiced artificially with two different secretory peptides and promoters to form a artificial operon and integrated into Bacillus subtilis A164; integration sites are the positions at which alkaline protease aprE and amylase gene amyE locate respectively; in addition, by the plasmid expression of Cre recombinase , resistance gene integrated into A164 is knocked down; pullulanase fermentation enzyme activity of pullulanase recombinant production bacterial strain CNP2 exceeds 1000 U / mL.

Description

technical field [0001] The invention belongs to the technical field of constructing safe production strains of enzymes used in the sugar industry, and relates to a method for constructing a high-yield pullulanase recombinant Bacillus subtilis. Background technique [0002] Pullulanase is an indispensable enzyme in the sugar production technology of the food industry. It is mainly a sugar chain debranching enzyme secreted and expressed by prokaryotic microorganisms. Type I pullulanase in this type of enzyme is often used in production and application, which can specifically cut the branched chain branch composed of α-1,6 glycosidic bonds in starch raw materials, so pullulanase and other starch hydrolysis The combined action of enzymes can completely enzymatically hydrolyze starch to obtain single-molecule glucose molecules, which has broad market prospects in food industries such as starch processing. In recent years, a lot of manpower and material resources have been invest...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/56C12R1/125
CPCC12N9/2457C12N15/75C12N2800/22C12Y302/01041
Inventor 纪明华俞峰刘校函王辉李统锋丁少明
Owner BAIYIN SINO BIOTECH
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