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Mannose

A mannanase and amino acid technology, applied in the directions of enzymes, hydrolases, enzymes, etc., can solve problems such as intolerance to pepsin, save food resources and breeding costs, improve utilization, and increase production efficiency.

Active Publication Date: 2013-03-27
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the mannanase produced by Trichoderma and Penicillium has acid resistance, it is not resistant to pepsin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cloning of Penicillium decumbens mannanase gene

[0026] 1.1 Extraction of total DNA

[0027] Cultivate Penicillium decumbens (P. decumbens) overnight, take an appropriate amount of cells into a centrifuge tube, centrifuge at 13,000 rpm for 5 min, discard the supernatant; add 400 μl of extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl, 1%SDS); then add 100mg of quartz sand or glass beads, shake vigorously in a bead beater for about 2min; after 20min in a water bath at 65°C, add 200μl 10M NH 4 AC, ice bath for 10min; centrifuge at 13000rpm for 10min, take the supernatant; add 2 times the volume of absolute ethanol, place at -20°C for 30min; centrifuge at 13000rpm for 10min, discard the supernatant; wash twice with 70% ethanol; air dry, add Dissolve in water and store at -20°C.

[0028] 1.2 Preparation of total RNA

[0029] The E.Z.N.A. Fungal RNA Kit of OMEGA Company prepared the mRNA of Penicillium decumbens, and its preparation process refers to...

Embodiment 2

[0037] Example 2 Construction of engineering bacteria whose expression sequence is SEQ ID NO: 3 mannanase gene

[0038] 2.1 Construction of Pichia pastoris engineered bacteria

[0039]The plasmid pT-Man2 was used as a template, and primers (agtgaattcCAGGTGGCGGAATATGGCC and agtgcggccgcTCAGATCGCAGCGACATG were used for PCR amplification. The PCR amplification conditions were 95°C for 4min; 94°C for 30S; 55°C for 40S; 72°C for 1.2min for 30 cycles; 72°C for 7min. After the product gel was recovered, Eco RI and Not I double enzyme digestion was performed first. Similarly, the expression plasmid pPIC9K was also subjected to Eco RI and Not I double enzyme digestion. The double enzyme digestion product, namely the cloned gene and the expression vector 4, was digested with T4 ligase. Ligated overnight at ℃. Finally, the ligated product was introduced into Escherichia coli BL21. The expression plasmid of the corresponding positive clone was named pPIC-Man, and the sequencing results sho...

Embodiment 3

[0049] Example 3 Fermentation and Enzymatic Properties Determination

[0050] 3.1 Shake flask fermentation of Pichia pastoris engineered bacteria

[0051] Inoculate Pichia pastoris CSD-2 into 5ml BMGY (1% yeast extract, 2% protein, 1.34% YNB, 4×10 -5 % biotin, 1% glycerol), cultivate overnight at 30°C, collect the bacteria by centrifugation, add the bacteria to 50ml BMMY induction medium (1% yeast extract, 2% protein, 1.34%YNB, 4×10 -5 % biotin, 0.5% methanol), add 50 μL methanol every 12 hours, induce culture for 5 days, take the supernatant (named PP-Man) for SDS-PAGE electrophoresis analysis, the results are as follows figure 1 shown. figure 1 The place indicated by the middle arrow is the recombinantly expressed mannanase, indicating that the mannanase whose sequence is SEQ ID NO: 3 of the present invention is expressed in the constructed Pichia pastoris engineering strain CSD-2.

[0052] 3.2 Shake flask fermentation of Trichoderma reesei engineered bacteria

[0053] T...

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Abstract

The invention relates to a novel mannose, an amino acid sequence of which is SEQ ID NO: 1. The mannose is respectively transferred to trichoderma reesei and pichia pastoris to form a recombined expression engineering strain. The invention provides a novel mannose gene, and respectively constructs pichia pastoris engineering bacteria and trichoderma reesei engineering bacteria for recombining and expressing the gene. The optimal action pH for generating the mannose by the pichia pastoris engineering bacteria and trichoderma reesei engineering bacteria is 4.5, and the optimal action temperature is 60 DEG C. The mannose provided by the invention is acid-resistant, heat-resistant, can be extensively used as a feed additive, and can effectively improve the utilization rate of the feed, so that the usage amount of feed in breeding is reduced, the gain resources and breeding cost are saved, and the productivity effect is increased.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a novel mannanase and a recombinant expression engineering bacterium for expressing the enzyme. Background technique [0002] Mannanase includes exonuclease, endonuclease and mannosidase, and the synergistic action of the three enzymes can completely degrade mannan. Among them, β-1, 4-D-mannanase (EC 3.2.1.78), also known as mannanase, belongs to endonuclease; it can hydrolyze β-1, 4-D-mannosidic bonds to generate Mannan oligosaccharides or mannan polysaccharides belong to the class of hemicellulase. Mannan is a linear polysaccharide linked by β-1, 4-D-mannopyranosidic bonds, if some residues in the main chain are replaced by glucose, or galactose is linked with mannose by α-1, 6-glycosidic bonds The residues are connected to form branches, which are called isomannans, mainly including galactomannan, glucomannan, and galactoglucomannan (Qi Junru et al....

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/19C12N1/15A23K1/165C12R1/84C12R1/885C12R1/80
CPCC12N9/2488C12R1/84C12N9/42C12R1/80C12N15/63C12R1/885A23K1/1653A23K20/189
Inventor 黄亦钧程斯达康丽华王华明曲音波刘国栋陈梅
Owner QINGDAO VLAND BIOTECH GRP
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