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Pullulanase with high secretion capacity and application thereof

A technology of pullulanase and high secretion, which is applied in the field of pullulanase and its application, can solve the problems of increasing industrial production costs, low extracellular expression level, difficult secretion, etc., to reduce the difficulty and cost of control, and reduce extracellular secretion The effect of high capacity and mild action conditions

Active Publication Date: 2019-04-16
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] However, because the pullulanase protein molecule is generally too large, it generally contains more than 900 amino acids, and the molecular weight of the single subunit is about 106kDa; in addition, the pullulanase single subunit usually contains 6 structural domains, and contains some unique Amino acid sequence, which causes pullulanase to easily form inclusion bodies, difficult to secrete extracellularly, and low level of extracellular expression
[0006] Therefore, so far, there are still many problems in the development of pullulanase at home and abroad. Among them, the low fermentation unit caused by insufficient soluble secretion of pullulanase is one of the key factors affecting its large-scale production
For example, after Deng Yi and others expressed the acid pullulanase derived from B. naganoensis in Bacillus, the enzyme activity was only 26.5U·mL -1 Zhen Jie et al. expressed the pullulanase gene derived from Anoxybacillus sp.LM18-11 in Bacillus subtilis, although the expression activity of the initial strain was improved, but the extracellular enzyme activity was only 42U. mL -1 Wu Lishuang et al. optimized the codon of the pullulanase gene from B.acidopullulyticus, then transferred it into Pichia pastoris for expression, and the final extracellular enzyme activity was only 0.4U·mL -1 , far below the requirements of industrial production
[0007] Moreover, the problem of low extracellular expression level of pullulanase will greatly increase the difficulty of separation and extraction of subsequent products during fermentation and production of pullulanase, further increase the cost of industrial production, and hinder the further popularization and application of pullulanase

Method used

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  • Pullulanase with high secretion capacity and application thereof
  • Pullulanase with high secretion capacity and application thereof
  • Pullulanase with high secretion capacity and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Extraction of the gene encoding pullulanase of the present invention and construction of recombinant bacteria containing the gene encoding pullulanase of the present invention

[0045] Specific steps are as follows:

[0046] (1) Extraction of Genomic DNA from Bacillus arzii CCTCC No:M 2017320

[0047] Pick a single colony of Bacillus arborii CCTCC No:M 2017320 and inoculate it into LB liquid medium, shake it at 37°C and 200rpm for 12 hours, then centrifuge at 12000rpm for 2 minutes to collect the bacteria; After washing the cells with water, centrifuge again to collect the cells and suspend the collected cells in 200 μL Tris-EDTA (trishydroxymethylaminomethane-ethylenediaminetetraacetic acid) buffer to obtain a resuspension; Add 20 μL of lysozyme to the solution, incubate at 37°C for 30 minutes, then add 5 μL of RNase, incubate at 37°C for 30 minutes, then add 30 μL of 10% SDS (sodium dodecyl sulfate) and 15 μL of proteinase K, and incubate at 37°C Incubate...

Embodiment 2

[0061] Example 2: Preparation of pullulanase from different sources and detection of pullulanase secretion ability from different sources

[0062] Specific steps are as follows:

[0063] (1) Preparation of pullulanase from different sources

[0064] The recombinant bacteria pulBac-PET20b(+) / E.coli BL21(DE3), pulBm-PET20b(+) / E.coli BL21(DE3) and pulBn-PET20b(+) / E.coli BL21(DE3) were inoculated into LB liquid medium, and cultured with shaking at 37°C for 8 hours to obtain seed liquid; then 2.5 mL of seed liquid was added to TB medium (containing -1 ampicillin), placed at 37°C, 200r·min -1 Shake culture under the conditions of 4h, add 0.4mM inducer IPTG, induce culture for 48h, and obtain the fermentation broth A and pulBac-PET20b(+) / E obtained from the fermentation of recombinant bacteria pul937-PET20b(+) / E.coli BL21(DE3) .coli BL21(DE3) fermentation broth B, pulBm-PET20b(+) / E.coli BL21(DE3) fermentation broth C, and pulBn-PET20b(+) / E.coli BL21(DE3) fermentation broth The f...

Embodiment 3

[0075] Embodiment 3: Separation and purification of pullulanase of the present invention

[0076] Specific steps are as follows:

[0077] The recombinant bacterium pul937-PET20b(+) / E.coli BL21(DE3) was inoculated into LB liquid medium and cultured for 10 hours to obtain seed solution; the seed solution was transferred to TB medium according to 5% inoculum size and fermented for 4 hours, Add 0.4mM inducer IPTG, induce culture 48h, obtain fermented liquid; Fermented liquid is at 4 ℃, 10000g centrifugation 20min collects the fermented supernatant that contains pullulanase; Supernatant adopts 70% (NH 4 ) 2 SO 4 Salt out and collect the precipitate by centrifugation; redissolve the precipitate in pH 6.8, 50mmol·L -1 The phosphate buffer was dialyzed for 20 hours, and the buffer was replaced once in the middle to obtain the sample; the sample was filtered through a 0.45 μm membrane to make the loading sample; the loading sample was purified by DEAE anion exchange chromatography c...

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Abstract

The invention discloses pullulanase with high secretion capacity and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. The pullulanase has high extracellular secretion capacity, and escherichia coli carrying the pullulanase is fermented in a shake flask for 48 hours, the total enzyme activity in the fermentation liquor can reach 212.0 U mL<-1>, wherein the extracellular enzyme activity reaches 200.5 U mL<-1>, which accounts for 94.5% of the total enzyme activity; therefore, the pullulanase is easy to prepare through fermentation, and the control difficulty and cost of the fermentation process can be obviously reduced; the pullulanase has mild action condition, and can hydrolyze alpha-1, 6-glucosidic linkage at a condition with a temperature of 40-60 DEG C and a pH value of 6.0-7.5, therefore, the pullulanase can reduce the cost in industrial transformation, and has potential utilization value in industries such as food, starch sugar and the like.

Description

technical field [0001] The invention relates to a pullulanase with high secretion capacity and application thereof, belonging to the technical fields of enzyme engineering and microbe engineering. Background technique [0002] Pullulanase, also known as pullulan-6-glucanohydrolase (pullulan-6-glucanohydrolase), is a kind of enzyme that can specifically cut pullulan polysaccharide (pullulan polysaccharide is composed of α-1, 4-glycosidic bond-linked maltotriose repeating units are polymerized by α-1,6-glycosidic bonds into a linear polysaccharide), amylopectin, and α-1,6 glucoside in branched dextrin hydrolase. [0003] Pullulanase is mainly divided into two types according to the difference in the type of glycosidic bond: type I pullulanase and type II pullulanase. Among them, type I pullulanase (EC 3.2.1.41) has the activity of hydrolyzing α-1,6-glucosidic bonds in pullulan polysaccharides and branched dextrins, but does not have the activity of hydrolyzing α-1,4-glucosid...

Claims

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Application Information

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IPC IPC(8): C12N9/44C12N15/56C12N15/70C12N15/75C12N15/81C12N1/21C12N1/19C12P19/16C12R1/19C12R1/07
CPCC12N9/2457C12N15/70C12N15/75C12N15/81C12P19/16C12Y302/01041
Inventor 段绪果沈镇炎张心怡赵林果张筠金璐陈佳琳
Owner NANJING FORESTRY UNIV
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