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Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant

A pullulanase and thermostability technology is applied in the field of pullulanase mutants and their preparation, and can solve the problems of low extracellular secretion efficiency and low extracellular secretion efficiency of recombinant pullulanase.

Active Publication Date: 2014-02-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The low efficiency of extracellular secretion of recombinant pullulanase may be determined by the enzyme's own amino acid sequence and spatial structure
Studies have shown that the molecular weight and structural complexity of proteins have a great impact on their soluble expression and secretion efficiency. Proteins with larger molecular weight and more complex structures are more likely to form insoluble protein aggregates, and the extracellular secretion efficiency is lower.

Method used

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  • Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant
  • Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant
  • Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Preparation of N-terminal truncation mutants of natural pullulanase

[0026] (1) Construction of N-terminal truncation mutants of natural pullulanase

[0027] Two N-terminal domain deletion mutants Puld1 and Puld2 of pullulanase derived from B.deramificans:

[0028] Based on the comparison and analysis of pullulanase sequences from different sources, the structure of the pullulanase protein from Bacillus demycotina was simulated and analyzed, and it was found that the pullulanase from Bacillus demycotina has six structural domains: CBM41, X25, X45, CBM48, A and C. Among them, the CBM41, X25 and X45 domains at the N-terminus of the protein are connected by a flexible linker, which has strong swingability; CBM41 belongs to the carbohydrate binding domain 41 family and has the function of binding to starch substrates, while the X25 and X45 domains Function unknown. However, the three structural domains of CBM48, A and C, are connected together in a very tight...

Embodiment 2

[0050] Example 2: Preparation of stacking mutants.

[0051] (1) Construction of superposition mutants

[0052] Two N-terminal domain deletion mutants D437H / D503Y / d1 and D437H / D503Y / d2 derived from the pullulanase double mutant D437H / D503Y of B.deramificans:

[0053] One domain of CBM41 at the N-terminus of the pullulanase double mutant D437H / D503Y was deleted and named as D437H / D503Y / d1; two domains of CBM41 and X25 at the N-terminus of the pullulanase double mutant D437H / D503Y were deleted and named as For D437H / D503Y / d2.

[0054] Preparation method of two N-terminal domain deletion mutants D437H / D503Y / d1 and D437H / D503Y / d2, the natural pullulanase coding gene of B.deramificans is replaced by the coding gene of pullulanase double mutant D437H / D503Y , other methods are the same as those of the N-terminal truncation mutant of natural pullulanase.

[0055] Preparation of mutants D437H / D503Y / d1 and D437H / D503Y / d2 encoding genes: Utilize rapid PCR technology, using expression v...

Embodiment 3

[0070] Example 3: This example illustrates an enzyme activity assay.

[0071] 1) Enzyme activity assay method

[0072] 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.

[0073] Enzyme activity assay steps:

[0074] A. Preheating: Take 2ml of 1.5% pullulan solution (50mM pH4.5 acetic acid buffer) in a test tube, put it in a 60°C water ...

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Abstract

The invention discloses a pullulanase mutant with improved secretion efficiency and heat stability and a preparation method of the pullulanase mutant, and belongs to the field of gene engineering and enzyme engineering. The secretion efficiency and the heat stability of pullulanase are improved through structural domain deletion mutation; deleted structural domains are CBM41, X45 and X25 structural domains or combinations thereof; and the mutation technical scheme capable of improving the secretion efficiency and the heat stability of pullulanase is provided. The obtained pullulanase structural domain deletion mutant has at least one of changed properties as follows: 1), the extracellular secretion efficiency is improved after recombinant bacteria are fermented; and 2), and the heat stability is improved under the conditions of pH 4.0-5.0 and 60 DEG C. Compared with natural pullulanase, structural domain deletion mutants are more suitable for production, preparation and applications of the pullulanase.

Description

technical field [0001] The invention relates to a mutant of pullulanase and a preparation method thereof, in particular to a technology for improving the secretion efficiency and thermal stability of pullulanase by using a protein engineering structural domain deletion method, which belongs to the field of genetic engineering and enzyme engineering . Background technique [0002] Pullulanase (EC3.2.1.41) is a starch debranching enzyme with important application value. It can hydrolyze α-1,6 in pullulan, pullulan, α-limit dextrin and other molecules. glucosidic bond. Pullulanase is suitable for acting on lower molecular weight dextrins, and its minimum action unit is maltosyl maltose, which is mainly used to prepare glucose by compounding with glucoamylase. Pullulanase can cut off the branch point in starch, accelerate the subsequent enzyme reaction, shorten the reaction time, increase the conversion rate of starch, and reduce the usage of other enzyme preparations for sacc...

Claims

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Application Information

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IPC IPC(8): C12N9/44C12N15/56C12N15/63
CPCC12N9/2457C12Y302/01041
Inventor 吴敬段绪果陈坚
Owner JIANGNAN UNIV
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