Pullulanase mutant and application thereof

A pullulanase, mutant technology, applied in the application, enzyme, hydrolase and other directions, to achieve the effect of good thermal stability

Active Publication Date: 2019-02-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, domestic research on pullulanase has gradually heated up, and many good research results have been achieved. However, there are still some problems in the development of high-activity, ...

Method used

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  • Pullulanase mutant and application thereof
  • Pullulanase mutant and application thereof
  • Pullulanase mutant and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: Recombinant bacteria construction

[0032] According to Anoxybacillus sp.WB42 (Wang J, Liu Z, Zhou Z. Cloning and characterization of a novel thermophilic amylopullulanase with a type Ipullulanase structure from Anoxybacillus sp.WB42[J]. 2018, 70 (5-6): 1700265.doi: 10.1002 / star.201700265) the base sequence of the pullulanase is shown in SEQ ID NO.2, and the cloning primers for the designed pullulanase are shown in Table 1 , The plasmid used to construct Escherichia coli is pET-22b(+), with Nde I, XhoI restriction sites. The pullulanase was cloned between the Nde I and Xho I restriction sites of the vector pET-22b(+) to obtain the recombinant plasmid pET-22b(+)-pulA. Proceed as follows:

[0033] (1) Design of pullulanase cloning primers;

[0034] (2) The gene pulA was cloned by PCR using Anoxybacillus sp.WB42 as a template;

[0035] (3) Recovering and purifying the above-mentioned PCR product, performing double digestion on the purified PCR product an...

Embodiment 2

[0037] Embodiment 2: the preparation of pullulanase mutant

[0038] (1) Single mutant

[0039] According to the gene sequence of pullulanase as shown in SEQ ID NO.2, primers for introducing mutant K419R were designed and synthesized, and fast PCR technology was used to carry the recombinant plasmid pET-22b encoding the gene of wild-type pullulanase (+)-pulA was used as a template, and the pullulanase gene was subjected to site-directed mutation, the DNA coding sequence was determined, and the 419th lysine codon was identified as an arginine codon, and the single mutant pullulanase K419R was obtained .

[0040] The site-directed mutagenesis primers for introducing the K419R mutation are:

[0041] The nucleotide sequence is the forward primer of SEQ ID NO.3:

[0042] 5'-GCATTCTCGATATTGACACGATG CGT GAAGTC-3' (the underline is the mutated base)

[0043] The nucleotide sequence is the reverse primer of SEQ ID NO.4:

[0044] 5'-ACAACTGCCTCGACTTC ACG CATCGTGTC-3' (the underl...

Embodiment 3

[0062] Embodiment 3: fermentation and purification of pullulanase

[0063] Pick the recombinant strain E.coli BL21(DE3) / pET-22b(+)-pulA expressing wild-type pullulanase and inoculate it in 5ml LB medium (containing 100μg / mL ampicillin antibiotic), 37℃, 200r / Incubate overnight with shaking. The overnight culture was inoculated into 100 mL LB medium (containing 100 μg / mL ampicillin antibiotic) at an inoculum size of 1% (v / v), and cultured at 37°C with shaking at 200 r / min until OD 600 At 0.6-0.8, add 10 μM inducer IPTG, induce at 16°C for 16-18 hours to obtain bacterial cells, and collect recombinant bacterial cells by centrifugation at 4°C and 8000r / min.

[0064] The above-mentioned recombinant cells collected by centrifugation were dissolved in 20 mL of binding buffer solution, ultrasonically disrupted, and after centrifugation at 13,000 g for 25 min, the supernatant was filtered with a 0.22 μm filter membrane. Then equilibrate the 1mL His TrapHP column with 10 times the co...

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Abstract

The invention discloses a pullulanase mutant and application thereof and belongs to the technical field of genetic engineering and enzyme engineering. According to the constructed pullulanase mutant,the enzyme activity of an obtained single mutant K419R (the 419th lysine is mutated to arginine) is 1.3 times higher than that of a wild mutant, and the remaining enzyme activity reaches 67.3 U/mg after the single mutant K419R is maintained at 67 DEG C for 30 minutes; the enzyme activity of an iterative mutant K419R+Y102C+K383C (the 419th lysine is mutated to arginine, and the 102th tyrosine and the 383th lysine are mutated to cysteine separately) is two times higher than that of the wild , the remaining enzyme activity of the iterative mutant reaches 88.3 U/mg after the iterative mutant is maintained at 67 DEG C for 30 minutes and is 43.1% of previous enzyme activity before heat preservation, and the thermal stability is high. The pullulanase mutant can be applied to a starch saccharification process.

Description

technical field [0001] The invention relates to a pullulanase mutant and application thereof, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Pullulanase is an α-1,6-glucosidic bond hydrolase, which can specifically and efficiently cut the branched chains in amylopectin. key enzymes. The starch saccharification process needs to be carried out at high temperature (60-65°C), and the saccharification time is generally 48-60h. Therefore, pullulanase must have the ability to maintain high enzyme activity and stability at high temperature. [0003] Glucose syrup produced by starch saccharification can not only produce crystalline glucose and high fructose syrup, but also serve as the main fermentation carbon source. Therefore, the development of pullulanase that can be compounded with glucoamylase is also particularly important at this stage. In recent years, domestic research on pullulanase has gradually heated up, ...

Claims

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Application Information

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IPC IPC(8): C12N9/44C12N15/56C12N15/70C12N1/21C12P19/14C12P19/04
CPCC12N9/2457C12N15/70C12P19/04C12P19/14C12Y302/01041
Inventor 周哲敏周丽崔文璟刘中美庞博
Owner JIANGNAN UNIV
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