A kind of production method and application of thermophilic fungus mannanase

A technology of mannanase and konjac gum, which is applied in the biological field to achieve the effects of good thermal stability, great application value, and rich variety

Active Publication Date: 2022-05-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are no literature reports and patent publications on the production of partially hydrolyzed konjac gum with high weight-average molecular weight using konjac gum as raw material

Method used

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  • A kind of production method and application of thermophilic fungus mannanase
  • A kind of production method and application of thermophilic fungus mannanase
  • A kind of production method and application of thermophilic fungus mannanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1, cloning and high-level expression of mannanase gene

[0081] 1. Cloning of the mannanase gene

[0082] Specific primers were used to amplify the mannanase gene from Cladosporium camphorii, and the sequences of the specific primers were as follows:

[0083] Upstream primer: 5′-ATGAAGTTATCATCCTTCGCT-3′;

[0084] Downstream primer: 5'-CTAGCTTGCGTTTCGAGTAGC-3'.

[0085] In the PCR reaction, the cDNA of Malbranchea cinnamonmea S168 was used as a template, and the above primer pair was used as primers, and Ex taq DNA polymerase (Takara Company) was used to amplify. The program was: pre-denaturation at 95°C for 5 minutes; 30 cycles of amplification at 94°C for 30s, 55°C for 30s, and 72°C for 1 min; total extension for 10 minutes. After the PCR product was detected by 1% agarose gel electrophoresis, it was recovered and connected to the pMD-18T vector (Takara Company), transformed into Escherichia coli by heat shock method, and a single colony was selected for ...

Embodiment 2

[0102] Embodiment 2, the purification of mannanase and the determination of enzymatic properties

[0103] 1. Purification of mannanase

[0104] Q-Sepharose FF (Q-Sepharose FF) ion-exchange chromatography: get 10mL of the fermented liquid fermented through the high-density fermentation of Example 1, obtain the supernatant through centrifugation at 10000rpm for 5min, in 20mmol / L Tris-HCl buffer ( pH 8.0) overnight, and loaded on a Q-sepharose column equilibrated with 20mmol / L Tris-HCl buffer (pH 8.0). Elute with 20mmol / L Tris-HCl buffer (pH8.0) for 20min, elute unbound protein, and linearly elute with 20mmol / L Tris-HCl buffer (pH8.0) containing 0-500mmol / L NaCl For protein, the elution time was 100min, and the eluate containing the target protein was combined and then dialyzed overnight with 20mmol / L MOPS buffer (pH7.5).

[0105] SDS-PAGE purification diagram as shown figure 2 shown. Wherein, swimming lane 1 is the fermentation supernatant, swimming lane 2 is the pure enzym...

Embodiment 3

[0132] Embodiment 3, hydrolysis different substrate concentration konjac gum

[0133] Weigh 100mL 50mmol / L MOPS buffer solution of pH7.5, add 1g, 5g, 10g, 20g, 30g konjac gum and stir well, add mannanase McMan5A according to the ratio of konjac gum to 200U / g, and place at 70°C It was hydrolyzed for 8 hours, and after enzymolysis, the reaction was terminated in a boiling water bath for 20 minutes to obtain an enzymolysis solution. The viscosity of the enzymolysis solution was measured at 25°C with a DV-1 rotational viscometer. The weight average molecular weight of the product was determined by gel exclusion chromatography. Dilute the product concentration to 10mg / mL with distilled water, centrifuge at 10000rpm for 5min, take the supernatant, pass through a 0.22μm filter membrane and load the sample, the chromatographic column is TSKgel GMPW XL , the column temperature was 35°C, the mobile phase was water, and the flow rate was 0.6mL / min. The molecular weight and molecular w...

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Abstract

The invention discloses a production method and application of mannanase. The method for producing mannanase provided by the present invention comprises: introducing the mannanase gene derived from Cladosporium camphoropsis S168 into recipient yeast to obtain recombinant yeast; performing basic fermentation culture, glycerol In the fed-feed fermentation stage and the methanol-feed-induced expression stage, the mannanase was obtained from the fermentation product. The mannanase expression level of the engineering bacteria of the present invention reaches 42200U / mL through high-density fermentation, and the optimal pH of the obtained enzyme is 7.5, and the optimal temperature is 75°C. It has acid and alkali resistance, good thermal stability, and excellent hydrolysis properties. Features, in addition, the present invention broadens the scope of application of mannanase, and the partially hydrolyzed konjac gum prepared by using this enzyme has great application value in industries such as food and feed, enriches the types of prebiotic products, and promotes the development of prebiotic products in my country. The development of yuan industry has great social and economic benefits.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a production method and application of thermophilic fungus mannanase. Background technique [0002] Mannanase (EC 3.2.1.78) is an endohydrolase that specifically hydrolyzes β-1,4-mannosidic bonds in mannan. According to the similarity of their amino acid sequences, mannanases can be divided into 5, 26, 113 and 134 families of glycoside hydrolases. As an important glycoside hydrolase, mannanase can hydrolyze different types of mannan (linear mannan, glucomannan, galactomannan) to produce mannan-oligosaccharides with a low degree of polymerization (Srivastava and Kapoor. Biotechnology Advances, 2017, 35:1-19). Therefore, mannanase has been widely used in various industries such as food and feed (Chauhan et al. Applied Microbiology and Biotechnology, 2012, 93: 1817-1830). Mannanase has a wide range of sources and is found in many microorganisms, plants and lower animals. Improving t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12P19/14A23L29/244A23L33/21A23K10/14A23K20/163A23K10/30C12R1/84
CPCC12N9/2494C12N15/815C12P19/14C12Y302/01078A23L29/244A23L33/21A23K10/14A23K20/163A23K10/30A23V2002/00A23V2250/5066A23V2250/5116
Inventor 闫巧娟李延啸江正强刘瑜杨绍青刘军
Owner CHINA AGRI UNIV
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