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Beta-amylase mutant with improved heat stability

An amylase and mutant technology, which is applied in the fields of genetic engineering and enzyme engineering, and can solve the problems of less molecular modification and no relevant literature reports.

Active Publication Date: 2017-09-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on microbial-derived β-amylase is currently mainly focused on the cloning, expression and enzymatic characterization of different hosts, but there are few molecular modifications for its enzymatic properties, and there is no relevant research on its thermal stability by directed evolution. Literature report

Method used

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  • Beta-amylase mutant with improved heat stability
  • Beta-amylase mutant with improved heat stability
  • Beta-amylase mutant with improved heat stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Construction of β-amylase mutant library based on error-prone PCR technology

[0019] Using error-prone PCR technology to introduce nucleotide mutations into the Bacillus flexus β-amylase gene. The reaction conditions for error-prone PCR are as follows:

[0020]

[0021]

[0022] The sequences of the upstream primer F and the downstream primer R are:

[0023] F: (5′-aa ccatgg cggtaaatggacagtcgtt-3′);

[0024] R: (5′-aa ctcgag ttaccaattattcgtatacgttg-3').

[0025] The PCR amplification procedure is: 94°C for 4min; 98°C for 10s, 53°C for 10s, 72°C for 1min 40s, 30 cycles; 72°C for 10min extension and incubation at 4°C.

[0026] The error-prone PCR product was recovered by agarose gel kit, the target gene was ligated with the vector pMD-18T to transform JM109. After cloning and culture, the target gene fragment was recovered with restriction enzymes Nco I and Xho I and passed through Nco I and Xho I. The digested expression vector pET24a(+) was connected and transform...

Embodiment 2

[0028] Example 2: Screening of β-amylase mutants with improved thermal stability

[0029] The Escherichia coli expressing parent β-amylase in pET24a(+) was used as the control bacteria.

[0030] Preliminary screening on LB agar starch plate: Recombinant strain E.coli BL21(DE3) / pET24a(+)-BFA was induced and expressed in TB fermentation medium for 48h, centrifuged to obtain extracellular supernatant, and 96 empty plates were obtained by high-throughput screening system The fermentation broth of each mutant was transferred to the LB agar starch plate one by one. Incubate overnight at 37°C for about 8 hours, coat the agar starch plate with iodine solution (0.03% w / v), observe the transparent circle, and select the mutant strain corresponding to the transparent circle that is significantly larger than the control.

[0031] Quantitative screening by DNS method in 96-well plate: select mutant strains with a higher transparent circle than the control, and quickly transfer the fermentation s...

Embodiment 3

[0032] Example 3: Enzyme activity analysis method

[0033] A β-amylase enzyme activity unit (1U) definition (U / mL): 1mL enzyme solution at pH 7.0, temperature 55 ℃, 1h hydrolyze soluble starch to produce 1mg maltose enzyme amount is called an enzyme activity unit.

[0034] Determination method: accurately draw 0.5mL of 2% soluble starch solution, place it in a 15mL colorimetric tube, add 0.4mL of 50mM pH 7.0 phosphate buffer solution, shake well, preheat in a 55℃ water bath for 5 minutes, accurately add 100μL of enzyme solution, and count immediately , Shake well, accurately heat the enzymatic hydrolysis reaction in a 55℃ water bath for 10 minutes, immediately add 1 mL of DNS, shake well, boil for 5 minutes, and cool in ice water. A reaction system with buffer solution instead of enzyme solution under the same conditions was used as a control. The above reaction system was added with 10 mL of distilled water, mixed well, and the absorbance was measured at a wavelength of 540 nm in ...

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Abstract

The invention discloses a beta-amylase mutant with improved heat stability, and belongs to the technical fields of genetic engineering and enzyme engineering. The beta-amylase mutant with improved heat stability is finally obtained through researching the orthogenesis of beta-amylase derived from Bacillus flexus and carrying out heat stability screening on parent beta-amylase by an error-prone PCR technology which is one of most frequently used methods. The half-life of the mutant Thr461Tyr / Asp506Asn / Asp519Lys at 55 DEG C is two times higher than that of wild beta-amylase, and the mutant always keeps high enzyme activity at 55-65 DEG C. The mutant is more suitable for being in the industrial application of starch sugar than the wild beta-amylase.

Description

Technical field [0001] The invention relates to a β-amylase mutant with improved thermal stability, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] β-Amylase is also called maltosidase and glycogen amylase, and its system is called α-1,4-glucan-4-maltohydrolase (α-1,4-Dglucan maltohydrolase), and the EC number is 3.2 .1.2. When acting on starch, the maltose is cut sequentially from its non-reducing end, and the hydrolyzed product is β-maltose, β-limit dextrin and a very small amount of β-glucose. β-amylase has considerable industrial application value. For a long time, barley β-amylase derived from plants has been widely used in many fields. For example, in the beer fermentation industry, it partially replaces barley malt for beer production, and in the food industry it is used to hydrolyze starch to produce maltose syrup and caramel. Since Higashihara et al. first determined that Bacillus megaterium (Bacillus megat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N15/56C12N15/70C12N1/21C12P19/12C12R1/19
Inventor 吴敬陈晟陈磊
Owner JIANGNAN UNIV
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