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Thermophilic recombinant II type pullulanase and application thereof

A technology of pullulanase and recombinant Escherichia coli, which is applied in the field of genetic engineering and can solve the problems of poor thermal stability and low catalytic efficiency

Active Publication Date: 2019-06-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently reported type II pullulanases generally have low catalytic efficiency and poor thermal stability.

Method used

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  • Thermophilic recombinant II type pullulanase and application thereof
  • Thermophilic recombinant II type pullulanase and application thereof
  • Thermophilic recombinant II type pullulanase and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1II

[0040] Cloning and recombinant expression of the type II pullulanase gene of embodiment 1

[0041] According to the suspected type II pullulanase gene (The Genbank accession number: WP_013906427.1) derived from the deep-sea hyperthermophilic and barophilic archaea Pyrococcus yayanosii CH1, the codon sequence of the gene sequence was carried out, and the optimized gene sequence (without The optimized gene sequence is almost not expressed in E. coli without a connexin tag.) SEQ ID NO.2 was synthesized by General Biosystems (Anhui) Co., Ltd., and connected to the expression vector pET-24a(+) Between BamH I and Xho I restriction sites, the recombinant plasmid pET-24a(+)-Pul Py . Next, we handed over the tag sequences Flag, Strep, Gst, Sumo, and Mbp to General Biosystems (Anhui) Co., Ltd. to synthesize them into pET-24a(+)-Pul Py Between the Nde I and BamH I restriction sites, the T7 tags of the above recombinant plasmids were replaced, and transformed into Escherichia coli BL21(DE...

Embodiment 2

[0044] Embodiment 2 recombinant type II pullulanase Sumo-Pul Py Determination of properties

[0045] 1. Recombinant type II pullulanase Sumo-Pul Py Activity analysis of

[0046] Recombinant type II pullulanase Sumo-Pul Py 3,5-dinitrosalicylic acid (DNS) method was adopted for the determination of the activity of the method, and the specific method was as follows: 100 μL reaction system (10 μL final concentration of 100 mM potassium phosphate buffer with different pH, 50 μL final concentration of 3% pullulan and 40 μL Enzyme solution) reacted at a certain temperature for 15 minutes, added 100 μL DNS reaction solution, boiled water bath for 6 minutes, cooled to room temperature with running water, diluted with distilled water, mixed, and measured OD 476 . One enzyme activity unit (U) is defined as the amount of enzyme required to release reducing sugar equivalent to 1 μmol of glucose reducing power per minute under the assay conditions.

[0047] 2. Recombinant type II pullu...

Embodiment 3II

[0063] Example 3 Type II pullulanase Sumo-Pul Py Analysis of the enzymatic properties of the truncation mutant △28N+△791C

[0064] First, the Pul Py The first 28 and 33 amino acids at the N-terminal and the amino acids after the 734, 784, 790, 1021, 1025, 1068, and 1077 amino acids at the C-terminal were removed to obtain 9 Pul Py The truncated gene sequences were synthesized into the vector pET-24a(+)-His+Sumo-Pul by General Biosystems (Anhui) Co., Ltd. Py Between the BamH I and Xho I restriction sites, the 9 new recombinant plasmids obtained were transformed into Escherichia coli BL21(DE3). The results showed that the specific enzyme activity of △735C at 95°C was Sumo-Pul Py 0.33 times that of Sumo-Pul. Py 2.02 and 2.31 times of that of Sumo-Pul Py There is also a certain degree of improvement ( Figure 9 ).

[0065] Pul PyThe first 28 amino acids at the N-terminal and the amino acids after the 790th position at the C-terminal were removed to obtain a combined trunc...

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Abstract

The invention discloses thermophilic recombinant II type pullulanase and application thereof, and belongs to the technical field of gene engineering. The thermophilic recombinant II type pullulanase is obtained through heterologous expression of II type pullulanase in escherichia coli, the most proper pH is 6.6, the thermophilic recombinant II type pullulanase has good pH tolerance under the condition that the pH value is 5.8-8.0, the most proper temperature is 95 DEG C, heat preservation is carried out for 10 h at 95 DEG C, the remaining enzyme activity is larger than 50%, and a relatively high specific activity can be shown under strong reduction conditions; if DTT is added into a culture environment, the specific activity of Sumo-PulPy can be increased by 237.2%. The invention also provides a combined truncated mutant delta28N+delta791C of the II type pullulanase Sumo-PulPy. The specific activity of the enzyme mutant is 32.18+ / -0.92 U / mg and is 5.99 times that of wild enzyme, and the mutant has important industrial application value and potential.

Description

technical field [0001] The invention relates to a thermophilic recombinant type II pullulanase and its application, belonging to the technical field of genetic engineering. Background technique [0002] Pullulanase (EC 3.2.1.41) is a starch debranching enzyme capable of hydrolyzing α-1,6 glycosidic bonds in amylopectin, pullulan, α-limit dextrin and β-limit dextrin. According to the hydrolysis method and product specificity, pullulanase can be divided into type I pullulanase and type II pullulanase. Type I pullulanase specifically hydrolyzes α-1,6 glycosidic bonds in pullulan and starch to produce maltotriose and linear oligosaccharides; type II pullulanase is a bifunctional enzyme that not only hydrolyzes α-1,6 glycosidic bonds in pullulan and amylopectin, have type I pullulanase function, and can hydrolyze α-1,4 glycosidic bonds in starch and related oligosaccharides by random endolysis , with α-amylase function. Pullulanase has important applications in syrup productio...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/56C12N9/44C12N15/70C12P19/14C12P19/00C12P19/04C12P7/48C12R1/19
CPCC12P7/48C12N15/70C12P19/14C12P19/04C12P19/00C12P19/16C12R2001/19C12N9/2457C12Y302/01041C12R2001/01C12N1/205
Inventor 周哲敏周丽崔文璟刘中美庞博
Owner JIANGNAN UNIV
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