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Method for acquiring novel pullulanase gene and high-yield strain and technology for producing enzyme

A technology of pullulanase and gene, which is applied in the field of genetic engineering and fermentation engineering, can solve the problem of high action temperature and achieve the effects of efficient preparation, high catalytic activity and easy acquisition

Active Publication Date: 2017-05-31
福建福大百特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lower pH (pH 4.5-5.0) of glucoamylase, the action temperature is higher (55 º C-65 º C), so the pullulanase used in conjunction with glucoamylase must also have similar enzymatic properties, in order to greatly expand its market and realize industrial application

Method used

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  • Method for acquiring novel pullulanase gene and high-yield strain and technology for producing enzyme
  • Method for acquiring novel pullulanase gene and high-yield strain and technology for producing enzyme
  • Method for acquiring novel pullulanase gene and high-yield strain and technology for producing enzyme

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Effect test

Embodiment 1

[0048] Embodiment 1—the synthesis of pullulanase gene from different sources

[0049] According to the accession number information in Table 1, each pullulanase gene sequence was downloaded from the NCBI database and synthesized.

[0050] Table 1 Pullulanase genes and their accession numbers

[0051]

Embodiment 2

[0052] Embodiment 2—Acquisition of novel pullulanase gene library

[0053] The pullulanase gene obtained in Example 1 was used as a template to clone the pullulanase gene obtained in different combinations. Use part of the bases in the relatively conserved sequence of the above-mentioned microbial pullulanase gene to form long primers in series, such as PULX1 and PULX2 in the appendix, under short-term PCR conditions [95°C 10min; 30×(94°C 30s; 56°C 1min; 72°C 0.1~2s); 68°C 10min] to amplify the target band. The obtained bands were cloned into the expression vector pHY-WZX to obtain the recombinant plasmid pHY-WZX-pulX. The obtained recombinant plasmid was preserved in Escherichia coli JM109. As a novel pullulanase gene library, it is used for the construction of subsequent secretory expression recombinant bacteria and the screening of pullulanase high-producing strains.

Embodiment 3

[0054] Embodiment 3—screening of novel pullulanase high-yielding bacterial strains

[0055] The above-mentioned recombinant plasmid was extracted by alkaline lysis to prepare a closed-circle plasmid and introduced into the host bacteria Bacillus licheniformis CBB302 by electric shock transformation method to obtain the corresponding recombinant bacteria; Great strain. The recombinant bacteria obtained from the primary screening were inoculated into the liquid pullulanase screening medium and cultured aerobically at 37 °C for 120 h to measure the enzyme production. The enzyme activity data are shown in Table 2. The strains with obvious transparent circles were stored in frozen storage tubes at -70°C after liquid culture for subsequent fermentation process establishment and efficient preparation of pullulanase.

[0056] Table 2 The formation of the transparent zone of the new pullulanase recombinant bacteria

[0057]

[0058]

[0059]

[0060]

[0061] Note: + / -, t...

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Abstract

The invention relates to a method for acquiring a novel pullulanase gene and a high-yield strain. The method comprises the following steps: adopting a system for comparing the structure similarity of more than ten pullulanase genes derived from different microorganisms; selecting high-conservative area for performing multi-sequencing combination and designing a long primer; adopting a short-time PCR technique and taking the chromosome DNAs from different microorganisms as templates, thereby acquiring a corresponding suspected pullulanase gene fragment; cloning the gene fragment into a high-expression vector pHY-WZX and performing heterologous expression on the bacillus subtillis; screening a slab by dibbing pullulan with the acquired converter; acquiring the converter with an obvious transparent ring, shaking flask and fermenting and then confirming the strain with the highest enzyme activity. A new process for efficiently fermenting pullulanase on the basis of special starch hydrolysate is established. The efficient preparation for pullulanase in 25L and 30m<3> fermentation system is realized. The highest enzyme activity can reach up to 880U / mL.

Description

technical field [0001] The invention provides a novel pullulanase gene, a method for obtaining a high-yield bacterial strain and a new technology for high-efficiency preparation of pullulanase, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] Pullulanase (EC 3.2.1.41) can specifically catalyze the hydrolysis of α-1, 6-glucosidic bonds of pullulan-type polysaccharides, making the branched chains of pullulan-type polysaccharides form a series of straight chains with different chain lengths. chain starch. The hydrolysis condition of the enzyme is that there are at least two α-1,4-glucosidic bonded glucose at both ends of the α-1,6-glucosidic bond. [0003] The hydrolysis properties of pullulanase determine that it has considerable value in improving the effect of amylase on starch, increasing the utilization rate of starch, reducing grain consumption, improving product quality and developing new products. For the starc...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/75C12N9/44C12N1/21C12R1/10
CPCC12N9/2457C12N15/75C12N2800/101C12N2800/22C12Y302/01041
Inventor 牛丹丹叶秀云
Owner 福建福大百特生物科技有限公司
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