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Novel pullulanase and coding gene and application thereof

A new pullulanase and gene technology, applied in the field of new pullulanase and its coding gene and its application, can solve the problems of few microorganisms and difficult microbial sampling

Active Publication Date: 2018-08-17
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, marine microorganisms, especially deep-sea microorganisms, are difficult to sample, and the culture conditions are special, and there are fewer microorganisms that can be isolated and cultured. In recent years, the establishment of a metagenomics technology system has made it possible to directly obtain enzyme resources from non-culturable microbial samples.

Method used

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  • Novel pullulanase and coding gene and application thereof
  • Novel pullulanase and coding gene and application thereof
  • Novel pullulanase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Cloning of Neopulluanase Neopullu31369 gene and construction of expression vector.

[0034] First, annotate and analyze the sequencing results of the metagenomic library of the deep-sea sediment sample E406 (water depth 3318, longitude 119°44.966'E, 18°44.922'N) to obtain the gene information of the new pullulanase; Gene sequence information, design specific primers, use the metagenomic library as a template, carry out PCR amplification, and obtain the Neopullu31369 coding gene fragment, the full length is 1767bp, which is consistent with the expected size (see figure 1 ). Such as figure 2 As shown, the recovered and purified Neopullu31369 gene PCR product was cut with XhoI and XbaI, and then ligated with the linearized vector pCold cut with XhoI and XbaI to form the expression vector pCold-Neopullu31369 containing the Neopullu31369 gene fragment.

[0035] The purified pCold-Neopullu31369 recombinant plasmid was sequenced bidirectionally with T7 and T7t pr...

Embodiment 2

[0050] Example 2: Recombinant expression and purification of neopullulanase Neopullu31369.

[0051] The recombinant expression plasmid with correct sequencing was transformed into the expression host strain BL21(DE3) to construct an engineering strain. Pick a single colony and inoculate it in Amp + In the LB liquid medium, 37 ℃ shaking culture overnight, as the seed fungus. First use 50% glycerol to preserve the seed, then take the remaining bacterial solution and inoculate it in fresh Amp at a volume ratio of 1:100. + In LB medium, shake vigorously at 37°C to amplify the culture until OD600 is about 0.6-0.8, add IPTG to a final concentration of 0.1mmol / L, and induce expression overnight at 18°C. Centrifuge at 4°C and 7500rpm for 10 minutes to collect the bacteria, and reserve 1ml of the bacteria liquid for SDS-PAGE analysis. After washing the cells with pre-cooled TE buffer (pH8.0), resuspend the cells with pre-cooled ultrasonic buffer. With 25% power, sonicate in an ice ...

Embodiment 3

[0052] Example 3: Determination of the enzyme activity of the new pullulanase Neopullu31369 on different substrates.

[0053] (1) DNS method

[0054] Take 2 test tubes, namely the control tube and the sample tube. Add 800 μl of Tris buffer solution and 100 μl of substrate solution to each of the two test tubes, incubate in a 37°C water bath for 5 minutes, then add 100 μl of inactivated purified enzyme solution (boiling in a boiling water bath for 10 minutes) to the control tube, and add 100 μl of purified enzyme solution to the sample tube , mixed immediately and timed, reacted accurately in a 37°C water bath for 30 minutes, then added an equal volume of DNS to terminate the reaction, developed color in a boiling water bath for 5 minutes, and measured the absorbance value with a 540nm spectrophotometer after cooling, repeated three times for each sample, and took the average value.

[0055] Enzyme activity unit (1U) is defined as the amount of enzyme required to catalyze the ...

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Abstract

The invention relates to novel pullulanase protein, a coding gene thereof and application of the protein serving as starch and pullulan hydrolase in the field of industry. A deep-sea sediment sample metagenome library is used as a study material, sequencing and gene function annotation analysis are performed on the metagenome library, a novel pullulanase gene is obtained by screening according toannotation information, a primer is designed according to gene sequence information, and metagenome is used as a template for PRC amplification to acquire a gene sequence coding novel pullulanase, wherein the DNA sequence of the novel pullulanase is shown as SEQ ID NO.1 in a sequence table. A target gene is inserted into a pCold expression vector for expression and purification to obtain polypeptide coded by the gene, and the amino acid sequence of the polypeptide is shown as SEQ ID NO.2 in the sequence table. Due to specific activity and enzymatic characteristics, the novel pullulanase can beapplied in industrial production related to pullulan, soluble starch and amylase hydrolysis.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a new pullulanase, its coding gene and its application. Background technique [0002] Neopullulanase is the most representative type of multifunctional amylase. In the process of producing sugar from starch, adding Neopululanase as an auxiliary enzyme can improve the conversion rate of sugar, and Neopullulan can also be used Enzymes to replace other transglycosidases, and use starch as raw material to produce prebiotics such as isomaltooligosaccharide and maltooligosaccharide. The current production of amylase and new pullulanase is low, and it is difficult to meet the requirements of the current market. Therefore, the development of a new type of new pullulanase has important research value and application in the starch processing industry, food, agriculture and pharmaceutical industries prospect. [0003] The ocean is rich in biodiversity, especially the unique enzyme syste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/44C12N15/55C12N15/11C12N15/70
CPCC12N9/2451C12N15/70C12Y302/01135
Inventor 王磊王金星
Owner SOUTH CHINA AGRI UNIV
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