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Preparation method of konjaku mannan oligosaccharide and special beta-mannase mutant adopted by same

A technology of mannanase and mannan oligosaccharides, which is applied in the preparation of konjac mannan oligosaccharides and β-mannanase mutants, which can solve the problems of high substrate concentration, low concentration of konjac fine powder, low concentration of konjac flour, etc. question

Active Publication Date: 2018-05-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese Patent Application No. 200910014349.X discloses a method for preparing mannan oligosaccharides by hydrolyzing konjac flour with β-mannanase derived from Bacillus, but the concentration of konjac flour used is very low, only 1%-1.5%, and its The time is as long as 30h; Chinese Patent Application No. 201310428885.0 discloses a preparation method of high-purity mannan oligosaccharides, but the concentration of konjac powder used in it is relatively low, which is 15%-25%; Chinese Patent Application No. 201510465107.8 discloses a A method for preparing konjac oligosaccharides by hydrolyzing konjac powder with acid mannanase, the concentration of the substrate (konjac powder) is also only 15%-25%
It can be seen that the above-mentioned patents are slightly insufficient in terms of practicability. At present, it is necessary to develop a high-efficiency production method of konjac mannan-oligosaccharides with high substrate concentration and short reaction time

Method used

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  • Preparation method of konjaku mannan oligosaccharide and special beta-mannase mutant adopted by same
  • Preparation method of konjaku mannan oligosaccharide and special beta-mannase mutant adopted by same
  • Preparation method of konjaku mannan oligosaccharide and special beta-mannase mutant adopted by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, construction of β-mannanase mutant gene

[0037] 1. Error-prone PCR

[0038] According to the wild-type gene of Rhizomucor miehei β-mannanase (Katrolia et al. Journal of Agricultural and Food Chemistry, 2013, 61:394-401), a pair of error-prone PCR primers without signal peptide was designed. The primer sequences are as follows:

[0039] Upstream primers:

[0040] 5'-CGCGGATCCGCTTCTTGGTTTGTCCAGACAAG-3';

[0041] Downstream primers:

[0042] 5'-CCGCTCGAGCTACTTCTTCTTGGCCATGGCATCAGC-3'.

[0043] Error-prone PCR system (50μL): 7mM Mg 2+ , 0.2mM Mn 2+ , 0.2 mM dATP, 0.2 mM dAGP, 1 mM dCTP, 1 mM dTTP, 0.2 μM RmMan5AF, 0.2 μM RmMan5AR, 2.5 U Taq DNA polymerase, 20 ng cDNA.

[0044] -5-

[0045] Error-prone PCR reaction conditions: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1.5 minutes, 30 cycles, and a total extension at 72°C for 5 minutes.

[0046] 2. DNA fragmentat...

Embodiment 2

[0063] Example 2, Preparation of β-mannanase mutants and determination of their enzymatic properties

[0064] 1. Induced expression of β-mannanase mutants

[0065] Inoculate the above recombinant Escherichia coli into liquid LB medium (containing 50 μg / mL kanamycin), shake culture at 37°C until the OD600 reaches between 0.6-0.8, add IPTG (final concentration 1mM), induce culture at 30°C Overnight, collect the bacteria at 10000×g, resuspend in buffer A (20mM phosphate buffer pH 8.0, 300mM NaCl, 20mM imidazole) and then sonicate, centrifuge at 10000×g for 10min, and collect the supernatant as the crude enzyme solution.

[0066] 2. Purification of β-mannanase mutants

[0067] The β-mannanase mutants were purified using an agarose Ni-IDA affinity column. Specific steps are as follows:

[0068] Equilibrate 5-10 column volumes of the Ni-IDA column with buffer A, load the above crude enzyme solution at a flow rate of 0.5mL / min, respectively use buffer A and buffer B (20mM phosphat...

Embodiment 3

[0091] Example 3, Pichia pastoris high-density fermentation expression of β-mannanase mutants

[0092] 1. Construction of recombinant bacteria

[0093] Using the β-mannanase mutant obtained in Example 1 as a template, PCR amplification was performed using the following primer pair to obtain a PCR amplification product. The primer sequences are as follows:

[0094] Upstream primers:

[0095] 5'-CCATGTACGTAGCTTCTTCGTTTGTCCAGACAAG-3';

[0096] Downstream primer: 5'-CCGCCTAGGCTACTTCTTGGCCATGGCATC-3'.

[0097] The PCR amplified product and the pGAPZαA vector were digested with restriction endonucleases SnaBI and AvrII, and the digested product was recovered and ligated to obtain a recombinant plasmid. The recombinant plasmid was transformed into Pichia pastoris GS115 to obtain recombinant bacteria B containing the recombinant plasmid.

[0098] 2. High-density fermentation

[0099] For the fermentation method, refer to the method in the document "Pichia Fermentation Process Gu...

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Abstract

The invention discloses a preparation method of konjaku mannan oligosaccharide and a special beta-mannase mutant adopted by the same. The beta-mannase mutant provided by the invention is from rhizomucor miehei, has the advantages of high stability, high enzymatic specific activity and the like and has very high application value in the industries of foods, feeds and the like. Engineering bacteriaformed by importing the beta-mannase mutant into pichia pastoris are fermented under a high density in a 5L fermentation tank, and the enzymatic activity of fermented liquid can reach 72,600U / mL (protein content is 9.1mg / mL). The beta-mannase mutant is used for hydrolyzing konjaku flour to prepare the konjaku mannan oligosaccharide; mainly mannan oligosaccharide with a polymerization degree beingbetween 2 and 6 is in hydrolysate; a konjaku hydrolysis rate is 90.2%; reducing sugar yield is 69.9%. The invention provides important bases for preparation of the konjaku mannan oligosaccharide by utilizing the konjaku flour.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a preparation method of konjac mannan oligosaccharide and a special β-mannanase mutant thereof. Background technique [0002] Mannan can be divided into four types according to the composition of monomers and connection methods, namely linear mannan, galactomannan, glucomannan and galactoglucomannan (Malgas et al. World Journal of Microbiology and Biotechnology, 2015, 31:1167-1175). Its main chain is composed of mannose (and glucose) connected by β-1,4-glycosidic bonds, and also contains side chains of galactose residues connected by α-1,6-glycosidic bonds. These mannans, as structural polysaccharides and energy storage polysaccharides, widely exist in the cell wall of plant tissues and the endosperm of dicotyledonous plants, such as: palm meal, coffee grounds and other agricultural wastes (linear mannan), seeds such as guar beans Endosperm (galactomannan), konjac tuber (glucomannan)...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12P19/14
CPCC12N9/2494C12P19/14C12Y302/01078
Inventor 闫巧娟李延啸江正强李斌王楠楠易萍游鑫
Owner CHINA AGRI UNIV
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