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Rhizomucor miehei-derived beta-1,3-glucanase and application thereof

A technology of glucanase and seeds, applied in the field of β-1,3-glucanase, to achieve good enzymatic properties and good application value

Active Publication Date: 2018-04-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Deposit date: June 21, 2011

Method used

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  • Rhizomucor miehei-derived beta-1,3-glucanase and application thereof
  • Rhizomucor miehei-derived beta-1,3-glucanase and application thereof
  • Rhizomucor miehei-derived beta-1,3-glucanase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Cloning of β-1,3-glucanase gene and expression in Pichia pastoris

[0043] Design upstream primer RmLam81AEcoRI (5'-attCCG GAATTC CAGAGTACAAGTGATGGAGACGAT-3', the underline shows the EcoRI restriction site) and the downstream primer RmLam81ANotI (5'-ATAAGAAT GCGGCCGC TCAGCGACGGAAGAAATGAGGAC-3', the underline shows the NotI restriction site), and the amino acid sequence of the encoded mature protein was amplified by PCR using the genomic DNA of Rhizomucor miehei CAU432 as a template. The amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 150 s, and 35 cycles; finally, extension at 72°C for 5 min. The product was recovered by 1% agarose gel electrophoresis and digested with EcoRI and NotI. Ligate the digested product with the same digested expression vector pPIC9K with T4 DNA ligase, transform E.coli DH5α competent cells by heat shock, spread on LB plates contai...

Embodiment 2

[0046] Example 2. High-density fermentation and purification of recombinant β-1,3-glucanase

[0047] 1. High-density fermentation of recombinant β-1,3-glucanase

[0048] Seed cultivation: Inoculate the recombinant Pichia strain A obtained in Experimental Example 1 into a 500mL Erlenmeyer flask filled with 100mL of BMGY medium, and vibrate at 30°C and 200rpm for 24-30h to obtain a seed solution with an OD600nm of about 3.0.

[0049] Glycerol fermentation stage: inoculate in 5L fermenter (2L BMGY culture medium is housed, solute is the material of following final concentration: the yeast extract that mass percent concentration is 1%, the peptone that mass percent concentration is 2%, 100mmol / L pH 6.0 phosphate buffer solution, the mass percent concentration is 1.34% YNB, and the mass percent concentration is 4×10 -5 % biotin, 1% glycerol by volume). Temperature in the process is 30 DEG C, adjust pH to 5.0 with ammoniacal liquor and phosphoric acid, control dissolved oxygen to...

Embodiment 3

[0055] Example 3. Properties of recombinant β-1,3-glucanase (RmLam81A)

[0056] 1. Optimum pH determination of RmLam81A

[0057] The optimal pH value of the enzyme was determined with the following four buffer systems with different pH values: acetic acid-sodium acetate buffer (pH4.0-6.0); citric acid-sodium citrate buffer (pH5.0-6.0); Phosphate buffer (pH6.0-7.5); Tris-HCl buffer (pH7.5-9.0). Then adopt the standard method to measure the enzyme activity of β-1,3-glucanase at 40 ℃, take the highest value of the enzyme activity as 100%, and calculate the relative enzyme activities measured at each pH respectively, the results are as follows image 3 shown. image 3 It was shown that the optimum pH value of recombinant β-1,3-glucanase (RmLam81A) was 5.5 (citric acid-sodium citrate buffer).

[0058] 2. Determination of pH stability of RmLam81A

[0059] Dilute the RmLam81A enzyme solution with the above four buffer solutions of different pH, keep the diluted enzyme solution in a...

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Abstract

The invention discloses rhizomucor miehei-derived beta-1,3-glucanase and application thereof. The rhizomucor miehei-derived beta-1,3-glucanase is thermophilic fungus rhizomucor miehei-derived endo-type beta-1,3-glucanase (RmLam81A) that specifically acts on the beta-1,3 glycosidic bond in curdlan polysaccharide. Pichia pastoris is used for heterologous expression, and after high-density fermentation culture, the final enzyme activity is up to 694U / mL. Food grade beta-1,3-glucan (the curdlan polysaccharide, laminarin, and the like) are used as raw materials for enzymatic hydrolysis to obtain beta-1,3-glucosyl-oligosaccharides with degree of polymerization (DP) of 2-5. The yield of the soluble oligosaccharides is about 75-85%. The beta-1,3-glucosyl-oligosaccharides can be obtained by slag liquid separation, material liquid enrichment and spray-drying of hydrolysate. The beta-1,3-glucanase disclosed in the present invention has excellent enzymatic properties. Compared with that of the chemical or enzymatic methods in the prior art for preparing the beta-1,3-glucosyl-oligosaccharides, the method using the rhizomucor miehei-derived beta-1,3-glucanase mild in reaction conditions, environmentally-friendly, and higher in the yield of the oligosaccharides, and has important application value in enzymatic preparation of the beta-1,3-glucosyl-oligosaccharides.

Description

technical field [0001] The invention relates to the field of food biotechnology, in particular to a β-1,3-glucanase derived from Rhizomucor miehei and an application thereof. Background technique [0002] β-1,3-glucanase (β-1,3-glucanase, EC 3.2.1.39), also known as laminarinase, is a specific hydrolysis of β-1 in β-1,3-glucan , 3-glycosidic enzymes. β-1,3-glucanase hydrolyzes glycans from the inner side of the sugar chain, and the products are a series of oligosaccharides of different sizes. β-1,3-glucanase is widely distributed, such as fungi, bacteria, plants and some lower animals and viruses, and has various biological functions (Ferrer, P. Microbial Cell Factories, 2006, 5:10). [0003] Microorganisms are the main source of β-1,3-glucanase. Bacterial β-1,3-glucanase mainly belongs to the glycoside hydrolase (GH) 16 family, and the secreted extracellular β-1,3-glucanase is mainly used to degrade the fungal cell wall in its growth environment Or plant-derived β-1,3-g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2405C12Y302/01039
Inventor 闫巧娟游鑫江正强杨绍青
Owner CHINA AGRI UNIV
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