Whole cell xylanase for promoting rumen fermentation, and preparation method thereof

A xylanase and whole-cell technology, applied in the field of genetic engineering, can solve the problems of complicated enzyme protein extraction and purification process, interference with digestive enzyme nutrient absorption, and restriction of popularization and application, so as to save dyeing and elution and ensure product specificity , to ensure the effect of amplification efficiency

Active Publication Date: 2014-01-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

In aquaculture production, because monogastric animals lack enzymes that degrade hemicellulose, xylan has almost no nutritional effect on monogastric animals, and undigested fiber will increase the viscosity of food, interfere with the action of digestive enzymes and nutrition The absorption of ruminants; due to the existence of rumen microorganisms, ruminants have a certain ability to degrade xylan, but in actual production, it is still necessary to supplement exogenous enzyme preparations to further improve the digestion efficiency of roughage for animals
Xylanase is a general term for a type of glycoside hydrolase that can specifically hydrolyze xylan into xylooligosaccharides and D-xylose. Due to its low expression level in natural materials, long production cycle, and enzyme protein extraction and purification process It is cumbersome and costly, which seriously limits its popularization and application

Method used

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  • Whole cell xylanase for promoting rumen fermentation, and preparation method thereof
  • Whole cell xylanase for promoting rumen fermentation, and preparation method thereof
  • Whole cell xylanase for promoting rumen fermentation, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0050] 1. Construction of recombinant plasmid pYD1 / ORF6-UN

[0051] Gene cloning strategy see figure 2 ,Specific steps are as follows:

[0052] ①Design primers containing two restriction enzyme sites, BamHI and XhoI.

[0053] Upstream primer (ORF6-BamHI):

[0054] 5'-TGACGGATCCGATTTTTGTCAAACTGCCGC-3'SEQ ID No.2,

[0055] Downstream primer (ORF6-XhoⅠ):

[0056] 5'-CACCTCGAGCGCCCCCTCGATATAGACCT-3'SEQ ID No.3,

[0057] ②PCR amplified xylanase coding gene ORF6-UN, using 50 μl amplification system (3 parallels).

[0058] Add the following components to the PCR tube:

[0059]

[0060]

[0061] Gently flick the PCR tube to mix, centrifuge slightly and place in a PCR machine.

[0062] The amplification conditions are: pre-denaturation at 94°C for 4 minutes; 6 cycle reactions (denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min); 24 cycle reactions (denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C 1min); 72°C...

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Abstract

The invention relates to the genetic engineering field, and concretely relates to a whole cell xylanase for promoting rumen fermentation, and a preparation method thereof. The whole cell xylanase for promoting rumen fermentation is characterized in that a gene cloning and yeast surface display technology is used to display a xylanase coding gene ORF6-UN from a Hu sheep rumen microorganism Fosmid library on the surface of Saccharomyces cerevisiae, and inducible expression, centrifuging, cell precipitate recovery and lyophilization are carried out to obtain dry cell powder which is ruminal whole cell xylanase; and the nucleotide sequence of the xylanase coding gene ORF6-UN is represented by SEQIDNo.1. The whole cell xylanase for promoting rumen fermentation can be supplemented to animal diets as a feed additive, and can increase the nutrition intake of animals and improve the fibrous substance utilization efficiency of the animals.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a whole-cell xylanase capable of promoting rumen fermentation and a preparation method thereof. Background technique [0002] Xylan is a common hemicellulose polysaccharide in plant cell walls, accounting for 1 / 3 of the total plant carbohydrates, second only to cellulose in content, and is the second most abundant available resource in nature. In aquaculture production, because monogastric animals lack enzymes that degrade hemicellulose, xylan has almost no nutritional effect on monogastric animals, and undigested fiber will increase the viscosity of food, interfere with the action of digestive enzymes and nutrition The absorption of ruminants; due to the existence of rumen microorganisms, ruminants have a certain ability to degrade xylan, but in actual production, it is still necessary to supplement exogenous enzyme preparations to further improve the digestion efficiency of a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42A23K1/165
CPCA23K20/189A23K50/10C12N9/248C12N15/81
Inventor 王佳堃杜文刘建新
Owner ZHEJIANG UNIV
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