Pullulanase mutant and preparation method thereof

A pullulanase and mutant technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, DNA preparation, etc., can solve the problems that thermal stability cannot fully meet the saccharification process, low catalytic efficiency, etc.

Active Publication Date: 2014-01-01
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the optimal temperature and thermostability of this enzyme cannot fully meet the needs of the saccharification process, and the catalytic efficiency is low

Method used

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  • Pullulanase mutant and preparation method thereof
  • Pullulanase mutant and preparation method thereof
  • Pullulanase mutant and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Recombinant bacteria construction

[0021] According to the pulA gene sequence registered on NCBI (NCBI accession number: AX203845), the starch debranching enzyme gene sequence pulA was synthesized by chemical total synthesis. The plasmid used to construct the expression vector in E. coli is pET20b(+) with T7 promoter. The pET20b(+) plasmid and the plasmid containing the pulA gene were subjected to NcoⅠ and HindⅢ double enzyme digestion respectively. After the digestion products were recovered by tapping the rubber, they were ligated with T4 ligase, and the ligated products were transformed into E.coli JM109 competent cells and cultured at 37°C At 8 hours, the transformants were picked and cultured with shaking in LB containing 100 mg / L ampicillin liquid, the plasmid was extracted, and the expression plasmid pulA / pET20b(+) was obtained for enzyme digestion verification.

[0022] The plasmid pulA / pET20b(+) was transformed into E.coli BL21(DE3) host bacteri...

Embodiment 2

[0023] Example 2: Preparation of mutants.

[0024] (1) Single mutation

[0025] Six single mutant enzymes D437H, D503R, D503F, D503W, D503Y and E589Y of pullulanase derived from B.deramificans:

[0026] Based on the comparison and analysis of pullulanase sequences from different sources, the structure of the pullulanase protein from Bacillus demycotina was simulated and rationally analyzed, combined with the results of the thermodynamic analysis of pullulanase, the debranching Bacillus spp. Three amino acid sites (Asp437, Asp503 and Glu589) in the Bacillus pullulanase molecule that have potential effects on the thermostability of the enzyme.

[0027] The aspartic acid (Asp) at position 437 in the pullulanase gene was mutated into histidine (His), named D437H; the aspartic acid (Asp) at position 503 in the pullulanase gene was ) were mutated into arginine (Arg), phenylalanine (Phe), tryptophan (Trp) or tyrosine (Tyr) respectively named D503R, D503F, D503W and D503Y; The glut...

Embodiment 3

[0078] Example 3: This example illustrates an enzyme activity assay.

[0079] 1) Enzyme activity assay method

[0080] The enzyme activity of pullulanase was determined by 3,5-dinitrosalicylic acid (DNS method). Under certain conditions, pullulanase catalyzes the hydrolysis of pullulan sugar to generate reducing sugar, and 3,5-dinitrosalicylic acid is reduced to a brownish-red amino complex after being heated together with the reducing sugar solution. The depth of its color within the range is proportional to the amount of reducing sugar, so the colorimetry can be performed at a wavelength of 540nm to calculate the enzyme activity. Definition of enzyme activity unit: Under the above conditions, the amount of enzyme that catalyzes the production of 1 μmol of glucose per minute is regarded as an activity unit.

[0081] Enzyme activity assay steps:

[0082] A. Preheating: Take 2ml of 0.5% pullulan solution (50mM pH4.5 acetic acid buffer) in a test tube and place it in a 60°C w...

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Abstract

The invention discloses a pullulanase mutant having a high specific activity and a high thermal stability, and a preparation method thereof. The mutant comprises one, two or three substituents relative to debranching Bacillus spp Pullulanase active amino acid residues and related with the thermal stability of pullulanase; and the active amino acid residues comprise 503th aspartic acid, and can be mutated to form arginine, phenylalanine and tryptophan or tyrosine, the above mutation improves the high specific activity and the high thermal stability of the pullulanase, and at least one of the cases comprising the rise of a best reaction temperature, the improvement of the thermal stability at a pH value of 4.0-5.0 and the improvement of the specific activity at a pH value of 4.0-5.0 is realized. The mutant is more suitable for the starch saccharification production process than wild pullulanase.

Description

[0001] This application is a divisional application with the patent number: 201210256804.9, the application date: July 23, 2012, and the patent name: a pullulanase mutant and its preparation method. technical field [0002] The invention relates to a mutant of pullulanase and a preparation method thereof, in particular to a technology for improving the specific activity and thermal stability of pullulanase by using a site-directed mutation method of protein engineering, and belongs to the fields of genetic engineering and enzyme engineering. Background technique [0003] Pullulanase (Pullulanase, EC3.2.1.41) is a debranching enzyme that can specifically cut α-1,6- Glycosidic bonds, used in the starch processing industry, can greatly improve the utilization rate and production efficiency of starch. Pullulanase is mainly used to prepare glucose by compounding with glucoamylase. Since the cutting efficiency of glucoamylase on the α-1,6-glucosidic bond in branched dextrin is ve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/44C12N15/56C12N15/10C12N15/70C12N15/75C12R1/07C12R1/125C12R1/10
CPCC12N9/2457C12Y302/01041
Inventor 吴敬陈晟段绪果陈坚
Owner JIANGNAN UNIV
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