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An optimized β-mannanase gene man and its expression vector in Pichia pastoris

A mannanase and gene technology, applied in the field of genetic engineering, can solve problems such as reducing feed utilization and affecting the contact between nutrients and digestive juices

Active Publication Date: 2015-12-02
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In beans, grains and their by-products, there is an anti-nutritional factor --- β-mannan, which has strong water absorption and is easy to form a gel in the digestive tract of monogastric animals, affecting the relationship between nutrients and Exposure to digestive juices reduces feed utilization

Method used

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  • An optimized β-mannanase gene man and its expression vector in Pichia pastoris
  • An optimized β-mannanase gene man and its expression vector in Pichia pastoris
  • An optimized β-mannanase gene man and its expression vector in Pichia pastoris

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Experimental program
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Effect test

Embodiment 1

[0078] Example 1 β-mannanase gene optimization and expression vector construction

[0079] 1) β-mannanase gene optimization and synthesis

[0080] The original β-mannanase gene VMAN was derived from a strain of Aspergillus niger that was screened and isolated from the natural soil near the konjac root. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. VMAN was codon-optimized (without changing the encoded amino acid sequence) and its stability was improved and then cloned into the pUC57 plasmid to obtain the optimized β-mannanase recombinant plasmid pUC57-MAN, whose nucleotide sequence is SEQ ID NO. 3.

[0081] 2) Digestion and ligation

[0082] Use EcoRI and NotI to digest the pUC57-MAN recombinant plasmid and Pichia pastoris expression vector pPICZαA, use NEB’s T4 ligase to connect the two to construct pPICZαA-MAN, and transform the ligated product into E. Culture and screen positive clones. Plasmids of positiv...

Embodiment 2

[0083]High-efficiency expression of the optimized β-mannanase recombinant strain of embodiment 2

[0084] The above-mentioned recombinant expression vector pPICzαA-MAN was linearized with SacI, and the linearized recombinant vector was electroporated to transform Pichia competent cells X33 to obtain Pichia recombinant strain X33 / MAN.

[0085] A single colony of the above-mentioned recombinant bacteria X33 / MAN was subjected to high-density fermentation culture. Configure 20L of basic salt medium, sterilize it in a 50L automatic control fermenter, and cool it to room temperature for later use. Adjust the pH value of the fermentation broth to 5.0 with ammonia water and phosphoric acid, control the dissolved oxygen to be greater than 30% by adjusting the rotation speed and air flow rate, and the fermentation temperature is 30°C. The entire fermentation process is divided into three stages: the first stage is the cell culture stage, inoculate the recombinant bacteria X33 / MAN-pPIC ...

Embodiment 3

[0086] Example 3 Activity analysis of the recombinant optimized β-mannanase

[0087] The reducing sugar produced by hydrolysis was determined by DNS method. The definition of each enzyme activity unit (U): Under the conditions of 55°C and pH 5.0, the amount of enzyme required to decompose β-mannan in locust bean gum per minute to produce a reducing ability equivalent to 1 μmol mannose. The experiment was repeated three times, and each sample was measured in three parallel experiments, and the relative error was controlled within 8%.

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Abstract

The invention relates to the field of genetic engineering and specifically relates to an optimized beta-mannase gene MAN and a pichia pastoris expression vector thereof. A beta-mannase gene with a nucleotide sequence as shown in SEQ ID NO.1 is modified to obtain an optimized gene of which the nucleotide sequence is as shown in SEQ ID NO.3. A recombinant pichia pastoris expression vector of the optimized beta-mannase gene is constructed; and the expression vector is transferred in a pichia pastoris competent cell X33 to obtain a trans-beta-mannase pichia pastoris bacterial strain capable of efficiently expressing beta-mannase by screening; an enzymatic property test indicates that the optimized beta-mannase gene can be stably and efficiently expressed and inherited in the pichia pastoris; enzyme activity and stability of the expressed beta-mannase are remarkably higher than those of the beta-mannase expressed in the original beta-mannase gene pichia pastoris.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an optimized β-mannanase gene MAN and a Pichia pastoris expression vector thereof. Background technique [0002] In beans, grains and their by-products, there is an anti-nutritional factor --- β-mannan, which has strong water absorption and is easy to form a gel in the digestive tract of monogastric animals, affecting the relationship between nutrients and Contact with digestive juices reduces feed utilization. β-mannanase is a kind of hydrolase that can specifically degrade β-mannan, and belongs to hemicellulase. The hydrolyzed substrates include galactomannan, glucomannan, galactoglucomannan and mannan, and the products include a small amount of monosaccharides and oligosaccharides composed of 2-10 monosaccharide molecules. It can effectively reduce the viscosity of the contents of the digestive tract; moreover, the mannan oligosaccharide produced by decomposing β-mannan ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/81C12N1/19C12R1/84
Inventor 聂金梅李阳源胡爱红钟开新周银华
Owner GUANGDONG VTR BIO TECH
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