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 problems such as inability to adapt to industrial applications, and achieve the effect of improving catalytic activity and thermal stability

Active Publication Date: 2014-11-05
无锡正元生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology improves how well an organism's own beta glucans are able to break down cell walls into smaller molecules called oligosaccharides or glycosaminogens (GAG). These small sugars help cells become better themselves when they die during their growth cycle. By comparing different types of bacteria found on Earth, this new method allows for faster production rates while maintaining high levels of GA content.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving beta gluconases' ability to resist heat stress during fermentative processing due to insurious factors like α-galactosidase and other carbohydrate hydroxamate compounds commonly associated therewith. These unwanted side effects include decreased bioavailability caused by excessive consumption of these sugars and impairments in taste quality when consumers drink them without any added sugar products. To address this issue, various methods were proposed including modifying certain parts of alpha-mannanase called endoinosporin A within each yeast species involved in beta-glacerbility.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Selection of mutation sites

[0030] Submit the deduced amino acid sequence of β-glucanase to the I-TASSER online server for homology modeling, and input the PDB file obtained from the modeling into Voronoia software to calculate the position of lysine in the structure. It can be seen from Table 1 that in the three-dimensional structure of β-glucanase, the average packing values ​​of all 12 lysines are between 0.47 and 0.62, all of which are on the surface of the protein and have the highest solution accessibility. This means that lysine located on the surface of the protein may have an effect on the thermostability of β-glucanase.

[0031] Table 1 The average packing density of 12 lysines in β-glucanase

[0032]

[0033] Using the genome of Bacillus tequila CGX5-1 as a template, the bglt gene encoding β-glucanase was amplified by PCR, and its nucleotide sequence was shown in SEQ ID NO.9. Using the overlap extension PCR technique and using the expression ve...

Embodiment 2

[0037] Example 2 Preparation of β-glucanase mutants K20S, K117S, K165S and K20S / K117S / K165S

[0038] (1) Site-directed mutation

[0039] 1) Construction of vector pET28a(+)-bglt

[0040] Using the genome of Bacillus tequila CGX5-1 as a template, the bglt gene was amplified by PCR. Primers are as follows:

[0041] Forward primer: 5'-C GGATCC ATGAAACGAGTGTTGCTAATT-3', the underline is the BamHI restriction site,

[0042] Reverse primer: 5'-T CTCGAG gTATTTTTTTGTATAGCGCAC-3', the underline is the XhoI restriction site, and the lowercase letter is the mutation site;

[0043] The PCR reaction system is: 5U / μL rTaq 1μL, 10×rTaq Buffer 5μL, 2.5mM dNTPs 4μL, 100μM forward primer 1μL, 100μM reverse primer 1μL, second-step PCR product 20μL, add double distilled water to make up to 50μL;

[0044] PCR reaction amplification conditions: pre-denaturation at 94°C for 5 minutes; followed by 35 cycles of 94°C for 1min, 56°C for 50s, and 72°C for 50s;

[0045] The PCR amplified product ...

Embodiment 3

[0070] Example 3 Analysis of Enzyme Activity and Protein Concentration

[0071] (1) Enzyme activity assay method:

[0072] 3,5-Dinitrosalicylic acid (DNS) method combined with improved AZO assay method for the determination of β-glucanase activity:

[0073] 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.

[0074] 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.

[0075]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 standa...

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Abstract

The invention discloses a 1,3-1,4-beta-glucanase mutant, and belongs to the fields of gene engineering and enzyme engineering. Lysine in the 20th position, 117th position and 165th position of 1,3-1,4-beta-glucanase from Bacillus terquilensis mutates through an overlapping extension PCR method to form serine in order to obtain single mutants K20S, K117S and K165S respectively. The above three mutation sites are integrally mutated to obtain a K20S/K117S/K165S triple mutant enzyme. Four mutant enzymes have higher catalysis activity and better thermal stability. Compared with wild enzymes, the above mutant enzymes are in favor of realizing the industrial application.

Description

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Claims

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Application Information

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Owner 无锡正元生物科技有限公司
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