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A kind of alkaline salt-tolerant pullulanase pula and its gene and application

A pullulanase and salt-tolerant technology, applied in the field of genetic engineering, can solve the problem of low alkaline pullulanase and achieve the effect of excellent properties and enhanced decontamination ability

Active Publication Date: 2019-09-13
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few research reports on alkaline pullulanase
In addition, there are few reports about the patent of alkaline pullulanase

Method used

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  • A kind of alkaline salt-tolerant pullulanase pula and its gene and application
  • A kind of alkaline salt-tolerant pullulanase pula and its gene and application
  • A kind of alkaline salt-tolerant pullulanase pula and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Pullulanase Gene PulA clone

[0046] Extraction of genomic DNA of halophilic and alkalophilic bacteria: a strain of alkaline pullulanase-producing microorganism was isolated from the bottom mud of Gan'an Dabusu Saline-alkali Lake in Jilin Province by using starch as the only carbon source screening medium, and tested by 16S identified as halophilic and alkalophilic bacteria. Genomic DNA of the halophilic and alkalophilic bacteria was extracted using the Bacterial Genome Extraction Kit (DP302) of Tiangen Company, and the specific operation was carried out in accordance with the instructions of the kit.

[0047] The degenerate primers Pul-F and Pul-R were designed and synthesized according to the conserved (HVRDFTSDP and FDMMGDHD) sequences of pullulanase genes:

[0048] Pul-F: 5'-GTNCGNGAYTTYACNTCNGAYC-3';

[0049] Pul-R: 5'-TCRTGRTCNCCCATCATRTC-3'

[0050] PCR amplification was performed using the above-mentioned total genomic DNA of halophilic and alkal...

Embodiment 2

[0055] Example 2 Preparation of recombinant pullulanase.

[0056] Introduced at the 5' and 3' ends of the gene, respectively Nco I and not I restriction site PulA-m- F and PulA-m- R is a pair of primers (see Table 1), and the genomic DNA of halophilic and alkalophilic bacteria is used as a template for PCR amplification. The PCR reaction parameters were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, extension at 72°C for 4 min, 30 cycles, and incubation at 72°C for 5 min. The expression vector pET22b(+) was double digested ( Nco I+ not 1), simultaneously the gene of coding pullulanase PulA double digestion ( EcoR I+ not 1), the cut gene fragment encoding pullulanase is connected with the expression vector pET22b (+), and the pullulanase gene is obtained PulA The recombinant plasmid pET22b- PulA And transform Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli strain BL21 / PulA.

[0057] Tak...

Embodiment 3

[0058] Example 3 Determination of the properties of recombinant pullulanase PulA

[0059] 1. Activity analysis of recombinant pullulanase

[0060] The activity of recombinant pullulanase was determined by 3,5-dinitrosalicylic acid (DNS) method: the specific method was as follows: at pH 9.0, 50°C, 1 mL of the reaction system included 100 μL of appropriate diluted enzyme solution, 900 μL substrate (1% beech pullulan), reacted for 10 min, added 1.5 mL DNS to terminate the reaction, and boiled water for 5 min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions.

[0061] 2. Determination of optimum pH and pH stability of recombinant pullulanase PulA

[0062] Optimum pH determination: The purified recombinant pullulanase PulA was subjected to an enzymatic reaction at 37°C in a 0.1M pH4.0–11.0 buffer. Determination of the pH stability of ...

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Abstract

The invention relates to the field of gene engineering, in particular to alkaline salt-tolerant pullulanase PulA and a gene and application thereof. The amino acid sequence of the alkaline salt-tolerant pullulanase PulA is as shown in SEQ ID No. 1, and the nucleotide sequence of the genome encoding gene PulA for encoding the pullulanase is as shown in SEQ ID No. 2. The invention further relates to a recombinant vector and a recombinant strain which contain the gene. The alkaline salt-tolerant pullulanase PulA has the advantages that the optimum pH of the pullulanase PulA is 9.0, and the residual enzyme activity of the pullulanase PulA when the pH is 10.0 is 77%; the optimum reaction temperature of the pullulanase PulA is 50 DEG C, the residual enzyme activity of the pullulanase PulA is 50% in the presence of 0.25-3.0M NaCl, and the pullulanase PulA is quite stable under 3M and 4M NaCl; the pullulanase PulA is alkaline, salt-tolerant and applicable to industrial fields such as detergent and food.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an alkaline salt-tolerant pullulanase PulA and its gene and application. Background technique [0002] Starch is a polymer compound formed by the polymerization of α-D glucose monomers through α-1,4-glycosidic bonds or α-1,6-glycosidic bonds. There are a lot of starch in animals and plants. All kinds of plants contain a high proportion of starch. Starch is a way for plants to store nutrients. Starch in plants is stored in the form of spherical granules and distributed in seeds, roots, tubers, etc. [0003] Amylase is a general term for a class of enzymes that can hydrolyze starch and glycogen. As a catalyst, amylase has been widely used in various industrial fields. Most plants, animals, and microorganisms contain amylase. Amylases are classified into α and β amylases according to the type of sugar isomers produced by the hydrolysis of the substrate. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/44C12N15/56C12N15/63C12N1/21C12N1/19C12N1/15C11D3/386C12R1/19C12R1/07
Inventor 王国增林娟叶秀云吴晶晶
Owner FUZHOU UNIV
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