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Acidic belta-mannase VMAN, gene thereof and application

A mannanase and acidic technology, applied in the field of genetic engineering, can solve the problems of low enzyme activity, unstable composition of enzymolyzed products, high production cost, etc., achieve good thermal stability, promote utilization rate, and reduce viscosity

Active Publication Date: 2011-04-06
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, the activity of β-mannan recombinase reported so far is generally low, the composition of enzymatic hydrolysis products is unstable, and the production cost is high

Method used

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  • Acidic belta-mannase VMAN, gene thereof and application
  • Acidic belta-mannase VMAN, gene thereof and application
  • Acidic belta-mannase VMAN, gene thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Isolation and cultivation of β-mannanase bacterial strain Aspergillus niger

[0077] A mold with mannanase activity was isolated from the natural soil near the konjac root, which was later identified as Aspergillus niger (preservation number: CGMCC No.4235)

Embodiment 2

[0078] Embodiment 2, the cloning of Aspergillus niger β-mannanase gene VMAN of Aspergillus niger

[0079] Use the RNA extraction kit to extract the total RNA of Aspergillus niger, and follow the reverse transcriptase SuperScript TM III Reverse Transcriptase Instructions for Synthesizing First Strand cDNA. Using cDNA as a template, design mannanase primers (VMAN5EcoRI, VMAN3NotI) for PCR amplification, perform double enzyme digestion on the PCR product with EcoRI and NotI, and then connect it to the Pichia pastoris expression vector pPICzalphaA that has undergone the same digestion. The product was transformed into Escherichia coli Top10 competent cells, and positive clones were obtained after screening with the antibiotic Zeocin. Plasmids of positive clones were extracted. The samples were sent to Shanghai Yingjun Biological Co., Ltd. for sequencing. The sequencing results showed that the cloned DNA insert contained the complete open reading frame of the β-mannanase gene. ...

Embodiment 3

[0084]Embodiment 3, the construction of the Pichia pastoris engineered bacterium comprising β-mannanase gene VMAN

[0085] The above-mentioned recombinant expression vector pPICzαA-VMAN was linearized with SacI, and the linearized recombinant vector was transformed into Pichia pastoris X33 by electroporation to obtain the recombinant Pichia pastoris strain X33 / VMAN.

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Abstract

The invention relates to the field of gene engineering and particularly provides belta-mannase which is derived from Aspergillus niger and has the collection of 4235 in the CGMCC and the amino acid sequence of SEQ ID NO.1. The invention also provides a gene of coding the belta-mannase, wherein the sequence of the gene is shown as SEQ ID NO.2. The optimal temperature of the mannose is 75 DEG C; the optimal pH value is 3.0; and the belta-mannose is acidic. Therefore, the anti-nutritional effect of the mannase is reduced effectively in an acidic environment of animal intestines and stomach, and the utilization rate of feed by the animals is increased. The invention also provides a method of effectively expressing the gene of the belta-mannase. The expressing quantity can achieve 11,785U / mL. Compared with the prior art, the invention ensures that expressing quantity of the belta-mannase is higher. Therefore, the industrial production cost can be largely reduced and the belta-mannase has greater application potential in fields of feed, food, petrochemical industry, brewing and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acidic β-mannanase and its gene and application. Background technique [0002] Mannan is a kind of plant hemicellulose ubiquitous in beans and cereals. It is a linear polymer connected by β-1,4-D-mannose and has high water absorption. The mannan contained in the feed absorbs a large amount of water in the digestive tract of monogastric animals, which increases the viscosity of the chyme, thereby resisting the peristalsis of the gastrointestinal tract, directly affecting the digestion and absorption of nutrients by the animals. In recent years, with the wide application of bean products (soybean meal, etc.) in feed, more and more attention has been paid to the anti-nutritional problem of mannan. Adding enzyme preparations to feed is the main way to solve this problem. The complete enzymatic hydrolysis of mannan requires the synergistic action of β-mann...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/42C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21C12N15/81C12R1/685
Inventor 张娟吴迪罗长财汪云飞陈丽芝谢建华
Owner GUANGDONG VTR BIO TECH
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