Pullulanase XWPu2 and gene thereof

A technology of pullulanase and gene, applied in the field of genetic engineering, can solve problems such as difficulty in mass production, low enzyme production of natural strains, and restrictions on popularization and application

Active Publication Date: 2013-03-13
KUNMING QACTIVE BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Pullulanase is an important industrial enzyme, but natural strains have low enzyme production, are difficult to produce in large quantities, and have high production costs, which limit its popularization and application

Method used

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  • Pullulanase XWPu2 and gene thereof
  • Pullulanase XWPu2 and gene thereof
  • Pullulanase XWPu2 and gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Pullulanase Gene wxya clone

[0034] Extraction of basophilic Paenibacillus ( Paenibacillussp.) Genomic DNA: CTAB lysis method, the specific steps are: centrifuge the fresh bacterial liquid cultured in liquid for 12 h, and add 800 μL solution Ⅰ (20 mM Tris, pH8.0, 2 mM Na 2 -EDTA, final concentration 20 mg / mL lysozyme), incubated at 37°C for 30 min, added 100 μL 10% SDS and mixed up and down, then added 10 μL 10 mg / mL proteinase K, incubated at 37°C for 30 min, added 150 μL μL 5 M NaCl and 150 μL 10% CTAB solution were mixed upside down, incubated at 65°C for 20 min, divided into 600 μL tubes, and then added 600 μL phenol / chloroform / isopropanol (volume ratio 25:24:1) For extraction, centrifuge at 12000 rpm for 10 min at 4 °C, take 300 μL of the supernatant and extract again in 300 μL chloroform / isopropanol (volume ratio 24:1) to remove impurities, and centrifuge at 12000 rpm for 10 min at 4 °C , then take 200 μL of the supernatant and add 2 times the vol...

Embodiment 2

[0041] Embodiment 2: Preparation of recombinant pullulanase XWPu2

[0042] pET -XWPu2 of Escherichia coli BL21(DE3) strain and pET-only - Empty plasmid for 28a(+) Escherichia coli BL21(DE3) strain was inoculated in LB (50 μg / mL Kan) medium with 0.1% inoculum, and shaken rapidly at 37 °C for 16 h. Then inoculate the activated bacterial solution into fresh LB (50 μg / mL Kan) culture solution with 1% inoculum, and culture with rapid shaking for about 2–3 h (OD 600 After reaching 0.6–1.0), add IPTG at a final concentration of 0.7 mM for induction, and continue shaking culture at 20 °C for about 20 h or at 26 °C for about 8 h. Centrifuge at 12000 rpm for 5 min to collect the cells. After suspending the cells with an appropriate amount of pH7.0 Tris-HCl buffer solution, the cells were disrupted by ultrasonic waves in a low-temperature water bath. The above intracellular concentrated crude enzyme solution was centrifuged at 13,000 rpm for 10 min, the supernatant was aspirat...

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Abstract

The invention discloses pullulanase XWPu2 and the gene thereof. The amino acid sequence of the pullulanase XWPu2 is shown in SEQID No. 1, and the nucleotide sequence of the pullulanase XWPu2 is shown in SEQID No. 2; and the recombinant vector and the recombinant strain of the pullulanase XWPu2 are provided. The pullulanase XWPu2 taking pullulanase as a substrate has the following properties that the optimum temperature is 50 DEG C; the optimum pH is 5.8-6.8; beta-mercaptoethanol having a final concentration of 1 mM, and metal ions Ca<2+>, Mn<2+>, Fe<2+> and K<+1> having a final concentration of 1 mM have a strong activation effect on enzyme; the residual enzyme activities after heat preservation for 140 minutes at 37 DEG C and at 50 DEG C are greater than 84% and greater than 52% in sequence; the residual enzyme activities after treatment for 90 minutes in a buffering solution having a pH of 5.0 and in a buffering solution having a pH of 8.6 are greater than 74% and greater than 57% in sequence; and the specific activity is 383.5 U/mg. Additionally, a qualitative analysis adopting thin-layer chromatography indicates that the pullulanase XWPu2 can be used for thoroughly hydrolyzing pullulanase to generate single maltotriose, hydrolyzing corn amylopectin to generate dextrin, and hydrolyzing cyclodextrin to generate glucose. The researches indicate that the pullulanase XWPu2 has a potential application prospect in the industries such as food, chemical industry, pharmacy and energy.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to type I pullulanase XWPu2 derived from alkalophilic Paenibacillus XW-6-66 and its gene. Background technique [0002] Pullulanase (Pullulanase, E C 3.2.1.41) is α-dextrin-6-glucan hydrolase, which can specifically hydrolyze the α-1,6-glucosidic bond of polysaccharides, making the branches of branched polysaccharides Chain debranching, forming a series of amylose with different chain lengths (Yu B, Wang J P, Zhang H X, et al . Molecules, 2011, 16:3010-3017). In the process of starch hydrolysis, pullulanase can be used in combination with α-amylase and glucoamylase to completely degrade starch into glucose; when used in combination with β-amylase, it can completely convert starch into maltose, which plays an important role in starch processing. important role. In addition, pullulanase and other glycoside hydrolases can synergistically act on different types of substr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/44C12N15/56C12N15/63C12N1/21C12N1/19C12R1/19C12R1/01
Inventor 黄遵锡
Owner KUNMING QACTIVE BIOLOGICAL TECH CO LTD
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