1,3-1,4-beta-glucanase mutant

A technology of dextranase and mutants, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems that thermal stability cannot be adapted to industrial applications, and achieve the effect of improving thermal stability

Active Publication Date: 2016-06-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the thermal stability of the modified enzyme BglTM ...

Method used

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  • 1,3-1,4-beta-glucanase mutant
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  • 1,3-1,4-beta-glucanase mutant

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Embodiment 1

[0026] Iterative saturation mutation of embodiment 1β-glucanase

[0027] Iterative saturation mutation was carried out at four positions 40, 43, 46 and 205 and the thermostability parameters of the mutants were determined. Using the plasmid pET28a(+)-BglTM (NiuC, ZhuL, ZhuP, LiQ.2015.Lysine-BasedSite-DirectedMutagenesisIncreasedRigidβ-SheetStructureandThermostabilityofMesophilic1,3–1,4-β-Glucanase.JournalofAgriculturalandFoodChemistry63:5249-5256) as a template, The 43rd, 46th and 205th positions introduced NNK degenerate codons (N: Ade / Cyt / Gua / Thy; K: Gua / Thy) by the Quickchange method to replace the target amino acids.

[0028] The site-directed mutagenesis primers for introducing the NNK degenerate codon at position 40 are:

[0029] Forward primer: 5'-cgtggcgggctaataacgtaNNKatgacgtcattgggtgaaatgc-3', capital letters are mutant bases,

[0030] Reverse primer: 5'-gcatttcacccaatgacgtcatMNNtacgttattagcccgccacg-3', capital letters are mutant bases;

[0031] The site-directed ...

Embodiment 2

[0044] Example 2 Induction, expression and screening of highly thermostable β-glucanase

[0045] (1) Induction and expression of β-glucanase

[0046] Pick a single colony from the plate and insert it into a 96-well plate containing 250 μL LB medium (containing 50 μg / mL kanamycin sulfate). Each well corresponds to a specific transformant and a wild-type clone is inoculated in each 96-well plate as a negative control. The obtained 96-well plate was placed in a shaker at 37° C. and cultured at 200 rpm for 11-12 hours. Use a row pipette to draw 20 μL of the cultured bacterial solution and transfer it into a 96-well plate containing 250 μL of fresh LB medium, and store the remaining bacterial solution at 4°C for temporary storage. After culturing at 37°C and 200rpm for 4 hours, IPTG and α-lactose with final concentrations of 0.06mM and 8mM were added to each well respectively and the culture temperature was lowered to 24°C for 6 hours. The OD of the obtained bacterial solution w...

Embodiment 3

[0051] Embodiment 3 Enzyme activity and protein concentration analysis

[0052] (1) Enzyme activity assay method:

[0053] 3,5-Dinitrosalicylic acid (DNS) method combined with improved AZO assay method for the determination of β-glucanase activity:

[0054] Enzyme activity definition: 1 mL of enzyme solution under the conditions of 40°C and pH value of 6.5, the amount of hydrolyzing β-glucan per minute to produce glucose reducing substances equivalent to 1 μmol is 1 enzyme activity unit, expressed in U / mL.

[0055] Determination of the enzyme activity of the fermentation broth: after the fermentation broth is centrifuged, the supernatant is diluted to an appropriate multiple to measure its enzyme activity.

[0056] Drawing of glucose standard curve: draw 1% glucose standard solution 2.0, 3.0, 4.0, 5.0, 6.0mL respectively into 50mL volumetric flask, dilute to the mark with distilled water, and make each milliliter contain glucose 200, 400, 600, 800 , 1000, 1200μg dilute stand...

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Abstract

The invention discloses a 1,3-1,4-beta-glucanase mutant and belongs to the field of gene engineering and enzyme engineering.40-bit serine and 43-bit serine, 46-bit glutamic acid and 205-bit histidine of 1,3-1,4-beta-glucanase from bacillus terquilensis CGX 5-1 are mutated into glutamic acid, glutamic acid, praline and praline respectively through an iterative saturation mutation method, and finally four strains of single mutants and three strains of compound mutants are obtained.Seven strains of mutate enzyme all represent better heat stability, and particularly S40E/S43E/E46P/H205P mutate enzyme has extremely good heat stability.Compared with wild enzyme, the mutate enzyme can be used in the industry more easily.

Description

technical field [0001] The invention relates to a 1,3-1,4-beta-glucanase mutant and belongs to the fields of genetic engineering and enzyme engineering. Background technique [0002] β-glucan is a non-starch polysaccharide found in the cell wall of grass plants. It is composed of thousands of β-D-glucose residues arranged linearly through β-1,3 or β-1,4 glycosidic bonds, and has a very high molecular weight. It can be dissolved in water, and the resulting solution has a high viscosity, which brings many disadvantages to the beer industry and the feed industry. Malt, the main raw material of the beer industry, contains a large amount of β-glucan. Failure to degrade β-glucan in wort will lead to excessive viscosity of wort, resulting in difficulty in filtration, prolonging the filtration time of wort mash, and reducing extracts. Content, for the finished beer will affect its non-biological stability. In the production process of pure draft beer, the presence of too much β-g...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12C5/00C12C7/04A23K20/189A23L29/00C12R1/19
Inventor 李崎钮成拓朱林江王金晶李永仙郑飞云刘春凤
Owner JIANGNAN UNIV
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