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Heat-resistant neutral pullulanase mutant and application thereof

A pullulanase and mutant technology, which is applied in the field of mutants of thermostable neutral pullulanase, can solve the problems of poor activity and stability of wild-type pullulanase, and achieves improved thermostability and secretion. The effect of improved efficiency and improved thermal stability

Active Publication Date: 2021-08-17
宿迁市江南大学产业技术研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to improve the thermal stability of neutral pullulanase and solve the problem of poor activity and stability of wild-type pullulanase under high temperature conditions

Method used

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  • Heat-resistant neutral pullulanase mutant and application thereof
  • Heat-resistant neutral pullulanase mutant and application thereof
  • Heat-resistant neutral pullulanase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Construction of recombinant plasmids

[0038] (1) Codon-optimizing the pullulanase gene from Bacillusthermoleovoran shown in SEQ ID NO.2 according to the codon preference rule of Escherichia coli, and the optimized nucleotide sequence is shown in SEQ ID NO.3;

[0039] (2) Use NcoI endonuclease and XhoI endonuclease to linearize the expression vector pET28a, and utilize NcoI endonuclease and XhoI endonuclease to double pullulanase gene (Btpul) shown in SEQ ID NO.3 Enzyme digestion treatment; the product after enzyme digestion treatment passed T 4 DNA ligase was connected to obtain wild-type pullulanase recombinant plasmid; the recombinant plasmid was transferred into E.coliJM109 competent cells, and the plasmid was extracted from positive transformants (plasmid extraction kit was purchased from OMEGA Company) to obtain wild-type Btpul recombinant plasmids.

[0040] (3 using PCR enzyme (purchased from TaKaRa company) adopts two-step PCR or whole plasmid PC...

Embodiment 2

[0103] Embodiment 2. Construction of recombinant bacteria

[0104] 1. Wild-type pullulanase expression strain: preparation of competent cells: referring to the standard operation on the Competent CellPreparation kit (purchased from TaKaRa Company), the preserved Escherichia coli was streaked and cultured to an LB-free plate, and picked Take the grown single colony to an LB test tube without antibiotics, and culture overnight at 37°C and 200rpm for 8-12 hours; after that, transfer to a 250mL Erlenmeyer flask containing 20% ​​LB antibiotic-free medium at an inoculum size of 2% , and the culture conditions were as described above at 37°C and 200 rpm for 8-12 hours overnight; 600 After about 0.6-0.8, put the 250mL Erlenmeyer flask on ice for 30min; divide the bacterial solution into sterilized 50mL centrifuge tubes in 25mL aliquots; centrifuge at 8000rpm for 5min at 4°C and discard the supernatant , add 10% solution A to gently resuspend the bacteria; centrifuge again at 4°C, 600...

Embodiment 3

[0109] Embodiment 3. Expression and purification of pullulanase

[0110] (1) Expression of wild-type pullulanase

[0111] The recombinant bacteria containing wild-type Btpul were inoculated into 5 mL containing kanamycin (50 μg·mL -1 ) in LB test tubes at 37°C and 200rpm for 8-12h; transfer the culture solution to 20% autoinduction medium (10g·L -1 Tryptone, 5.0g·L -1 Yeast extract, 10g·L -1 α-lactose, 5.0g·L -1 Glycerin, 1.0g·L -1 Glucose, 7.1g·L -1 Na 2 HPO 4 , 6.8g·L -1 K H 2 PO 4 , 2.67g L -1 NH 4 Cl, 0.71g L -1 Na 2 SO 4 and 0.25g·L -1 MgSO 4 ; pH7.5) in a 250mL Erlenmeyer flask, and add kanamycin to its final concentration of 50μg·mL -1 , cultivated at 37°C and 200rpm for 2-3h; lowered the temperature to 17°C, and continued to cultivate for 48-60h to express the wild-type recombinant protein.

[0112] (2) Expression of pullulanase mutants

[0113] The double-plasmid recombinant bacteria containing the recombinant plasmid of pullulanase mutant gene and...

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Abstract

The invention discloses a heat-resistant neutral pullulanase mutant and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the invention, amino acids at the 73th site, the 135th / 314th site, the 135th / 314th / 126th site and the 135th / 314th / 126th / 72nd site of pullulanase are respectively mutated, so that four pullulanase mutants with improved thermal stability and unchanged or improved catalytic efficiency are obtained. At the temperature of 70 DEG C, the half-life periods of the four mutants are respectively 2.6, 1.9, 3.3 and 3.1 times that of a wild type; and the Tm values are respectively increased by 7.0 DEG C, 6.5 DEG C, 6.7 DEG C and 6.9 DEG C. The extracellular enzyme activity of pullulanase reaches 9.7 U / mL by fusing OptPelB at the N terminal of the high-temperature-resistant neutral pullulanase mutant, and is 4.9 times that of a wild type. The heat-resistant neutral pullulanase mutant capable of secreting and expressing has the application potential of high-value utilization of starch raw materials.

Description

technical field [0001] The invention relates to a heat-resistant neutral pullulanase mutant and its application, belonging to the technical fields of enzyme engineering and genetic engineering. Background technique [0002] Pullulanase (EC 3.2.1.41) is well known for its specific starch debranching activity. Pullulanase belongs to the superfamily of glycoside hydrolases, which are capable of hydrolyzing α-1,6 glycosidic linkages in pullulan, dextrin, pullulan and other oligosaccharides. At present, pullulanase is mainly used in the saccharification of starch. Adding pullulanase in the starch saccharification process can reduce the amount of saccharifying enzyme and shorten the reaction time, thereby increasing the yield and conversion rate. Compared with debranching enzymes such as isoamylase, pullulanase can hydrolyze only two glucose linked by α-1,6 glycosidic bonds and two glucose linked by α-1,4 glycosidic bonds. chain segment. Therefore, pullulanase can maximize the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/44C07K19/00C12N15/62C12N15/70C12N1/19C12P19/16C12P19/04C12R1/19
CPCC12N9/2457C12Y302/01041C12N15/70C12P19/16C12P19/04C07K2319/02C12N2800/22
Inventor 聂尧穆晓清毕家华
Owner 宿迁市江南大学产业技术研究院
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