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Heat-resistant beta-mannanase and its coding gene, recombinant bacterium and use

A mannanase and recombinant bacteria technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of poor heat resistance, inability to resist the temperature of feed pelleting, limiting the use of β-mannanase, etc. Good acid stability, wide catalytic pH range and high specific activity

Active Publication Date: 2012-10-17
邳州市鑫盛创业投资有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The currently known β-mannanase has poor heat resistance and cannot resist the feed pelleting temperature, which limits the use of β-mannanase in feed

Method used

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  • Heat-resistant beta-mannanase and its coding gene, recombinant bacterium and use
  • Heat-resistant beta-mannanase and its coding gene, recombinant bacterium and use
  • Heat-resistant beta-mannanase and its coding gene, recombinant bacterium and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1, Discovery of thermostable β-mannanase (MANN-cx1 protein) and its coding gene

[0038] 1. Error-prone PCR amplification of β-mannanase gene

[0039] Using the optimized wild-type mannanase gene as a template, the error-prone PCR reaction system contains: dNTP (2.5mM), dCTP (10mM), dTTP (10mM), Mg 2+ (25mM), Mn 2+ (10 mM), Taq DNA polymerase (5 U). Primers were E: 5′-CCG GAA TTC TTG CCA AAG GC-3' (the underline is the restriction endonuclease EcoR I site), X: 5'-GC T CTA GA T TAA GCA GAA TC-3' (the underline is the restriction endonuclease XbaI cutting site). The PCR reaction conditions were: denaturation at 94°C for 1 min, annealing at 52°C for 30 s, extension at 72°C for 1 min, and 30 cycles. PCR products were detected by 1% agarose gel electrophoresis.

[0040] 2. Construction and screening of mutant expression library

[0041] The error-prone PCR product was digested with restriction endonucleases EcoR I and Xba I, and then connected to the self-r...

Embodiment 2

[0046] Embodiment 2, the construction of recombinant bacteria and the expression of thermostable β-mannanase (MANN-cx1 protein)

[0047] 1. Construction of engineering bacteria

[0048] 1. Synthesize the DNA (MANN-cx1 gene) shown in sequence 2 of the sequence listing.

[0049] 2. Using the MANN-cx1 gene synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0050] F1: 5'-CCG GAA TTC TTG CCA AAG GC-3' (underlined EcoR I restriction recognition sequence);

[0051] R1: 5'-GC T CTA GA T TAA GCA GAA TC-3' (Xba I restriction recognition sequence is underlined).

[0052] 3. The PCR amplified product of step 2 was double-digested with restriction enzymes EcoR I and Xba I, and the digested product was recovered.

[0053] 4. Digest the vector pPICZαA with restriction endonucleases EcoR I and Xba I to recover the vector backbone (about 3600 bp).

[0054] 5. Ligate the digested product of st...

Embodiment 3

[0083] Example 3, Enzymatic property analysis of thermostable β-mannanase (MANN-cx1 protein)

[0084] 1. Determination of the optimum pH value

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Abstract

The invention provides a heat-resistant beta-mannanase and its coding gene, recombinant bacterium and use. The heat-resistant beta-mannanase as a protein is shown in the formula (a) or (b). The heat-resistant beta-mannanase shown in the formula (a) comprises the amino acid sequence 1 of the sequence table; and the heat-resistant beta-mannanase shown in the formula (b) is a protein derived from the amino acid sequence 1 though replacement and / or deletion and / or addition of multiple amino acid residues and having a beta-mannanase activity. The invention also provides the coding gene of the heat-resistant beta-mannanase and the recombinant bacterium which is engineering bacterium and contains the coding gene. The recombinant bacterium is recombinant pichia pastoris. The heat-resistant beta-mannanase has a high specific activity, good acid stability and a wide catalysis pH range, and is suitable for being used as an additive of monogastric animals such as pigs and chickens.

Description

technical field [0001] The invention relates to a heat-resistant β-mannanase, its coding gene, recombinant bacteria and application. Background technique [0002] Mannan is an important component of plant hemicellulose, which is a linear polysaccharide linked by β-1,4-D mannopyranose. Mannan has high hydrophilicity, absorbs a large amount of water in the digestive tract of monogastric animals, increases the viscosity of the contents of the digestive tract, resists gastrointestinal peristalsis, and directly affects the digestion and absorption of nutrients by animals. The content of mannan in soybean meal is above 1.2%. In recent years, with the widespread application of bean products (soybean meal, etc.) in animal diets, the anti-nutritional effect of mannan has also attracted more and more attention. At present, my country's soybean import volume is more than 30 million tons, of which more than 80% are used for feed. [0003] The complete enzymatic hydrolysis of mannan r...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A23K1/165C12R1/84
Inventor 曹云鹤崔栩王春林陆文清李德发
Owner 邳州市鑫盛创业投资有限公司
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